Immunostimulatory compositions and methods

ABSTRACT

The invention provides conjugates comprising an immune co-stimulatory polypeptide and an antigen or infectious agent. The conjugates are useful for generating or enhancing an immune response against the antigen or infectious agent. The invention also provides immune cells modified with a conjugate that are useful for generating or enhancing an immune response to an antigen or infectious agent. The invention also provides immunostimulatory moieties comprising an immune co-stimulatory polypeptide that are useful for stimulating an immune response. The invention also provides immunotherapy methods and methods of treating or preventing infections.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.11/635,066, filed Dec. 7, 2006, which claims the benefit under 35 U.S.C.§119(e) of the filing dates of the following U.S. provisionalapplications: 60/748,177 (filed Dec. 8, 2005); 60/771,179 (Feb. 6,2006); 60/799,643 (filed May 12, 2006); and 60/863,173 (filed Oct. 27,2006). Each of the foregoing applications is incorporated by referenceherein in its entirety.

FIELD OF THE INVENTION

The present invention generally relates to methods of generating orenhancing an immune response, including an immune response against anantigen, to compositions for effecting the methods, and to modifiedimmune cells useful in such methods.

BACKGROUND OF THE INVENTION

Approaches directed to boosting a host's immune response to addressdiseases and conditions characterized by a deficiency in immunity orresolvable by a more aggressive immune response have been described.Exemplary diseases or conditions where such approaches may beadvantageous include cancer, influenza and human immunodeficiency virus(HIV).

Cancer treatments involving surgery, chemotherapy and radiotherapy arecommonly used, but these approaches lack tumor specificity, resulting inadverse side effects and less than satisfactory clinical responses.Accordingly, methods of boosting the immune response to cancer byspecifically directing that response to the target cancerous cellswithout significant, detrimental effects on normal cells would offerdistinct advantages over traditional cancer therapy.

There is a consensus that immune surveillance plays a role in theprevention and eradication of tumors, and adaptive immunity mediated byT cells plays a role in this process. See, e.g., Pardoll, Nat. Rev.Immunol. 2002, 2:227-38; Rosenberg, Nature 2001, 411: 380-84; Finn, O J.Nat. Rev. Immunol. 2003, 3:630-41. T cell-mediated immunity also plays arole in various immunotherapeutic approaches that have shown efficacy inpreclinical and limited clinical settings. See, e.g., Pardoll, supra;Finn, supra; Antonia et al., Curr. Opin. Immunol 2004, 16:130-6. Tumorsare targeted by the immune system because they express tumor associatedantigens (TAAs), which are either mutated or over/aberrantly expressedself-proteins, or proteins derived from oncogenic viruses. See, e.g.,Finn, supra; Antonio, supra. Under physiological conditions, tumorantigens are picked up by dendritic cells (DC), carried to peripherallymphoid organs, and presented to naïve T cells under immunogenicconditions allowing for their activation and differentiation intoeffector cells (T_(eff)). These cells then traffic to tumor sites andgenerate anti-tumor responses for tumor eradication. See, e.g., Spiotto,et al., Immunity. 2002; 17:737-47; Ochsenbein et al., Nature 2001;411:1058-64; Yu et al., Nat. Immunol. 2004; 5:141-9.

A productive T cell response requires three distinct signals: Signals 1,2, and 3. Signal 1 is generated by T cell receptors (TCR) interactingwith nominal peptides presented by major histocompatibility complex(MHC) molecules on the surface of professional antigen presenting cells(APCs). Signal 2 is mediated by a series of costimulatory molecules andis critical to a sustained immune response. Signal 3 is transduced viacytokines elaborated by activated lymphocytes and APCs, such asmacrophages and DC, and is important to the maintenance of effectorimmune responses.

Tumors have developed various mechanisms to evade immune surveillance.These mechanisms include: (i) lack of Signal 1, arising from either theinefficient display of MHC/tumor antigen bimolecular complexes on tumorcells, defects in the transduction of this signal, or expression of MHChomologues, MIC, that inhibit natural killer (NK cells) expressing NKG2inhibitory receptors; (ii) absence of Signal 2 originating from the lackof costimulatory molecules or expression of co-inhibitory molecules ontumor cells; (iii) tumor-mediated suppression of immune responsesthrough the secretion of anti-inflammatory molecules, induction ofanergy in tumor-reactive T cells, physical elimination of T_(eff) cellsvia apoptosis, or induction of naturally occurring CD4⁺CD25⁺FoxP3⁺ Tregulatory (T_(reg)) cells, and (iv) regulation of immunity by the tumorstroma. Accumulating evidence suggests that many of these mechanisms mayoperate simultaneously in patients with large tumor burdens.

Cancer vaccines which include antigens from the target cancer haveattracted particular interest because of the promise of specificity,safety, efficacy and the long-term memory of the immune system that mayprevent recurrence of the cancer. Once it was established that theimmune system plays an important role in safeguarding individualsagainst cancer and may be modulated to eradicate existing tumors inanimal models, intense efforts were devoted to the development oftherapeutic vaccines. See, e.g., Berzofsky et al., J. Clin. Invest 2004,113:1515-25; Platsoucas et al., Anticancer Res. 2003; 23, 1969-96; Finn,supra. Current vaccine strategies include the use of specific TAAs inconjunction with nonspecific or specific adjuvants, whole tumor celllysates, tumor cells genetically modified to express costimulatorymolecules, cytokines, and/or chemokines, DC pulsed with tumor antigensor transfected with tumor RNA or DNA, and intratumoral injection of arange of vectors encoding various immunostimulatory molecules. Thelimited efficacy of these approaches may stem from the ability ofprogressing tumors to control immune responses using one or severalimmune evasion strategies, or due to immunosuppressive mechanisms,inefficient presentation of TAAs, or lack of potent activation of DC,T_(eff) cells, and NK cells.

SUMMARY OF THE INVENTION

The present invention provides immunostimulatory compositions andmethods.

In accordance with one embodiment, the invention provides a combinationcomprising (a) a first conjugate comprising (i) a conjugate membercomprising a first immune co-stimulatory polypeptide and (ii) aconjugate member comprising a first member of a binding pair; and (b) asecond conjugate comprising (i) a conjugate member comprising a firstantigen and (ii) a conjugate member comprising a second member of thebinding pair. In one embodiment, the first member of the binding pairmay comprise avidin or streptavidin and the second member of the bindingpair may comprise biotin. In another embodiment, the first conjugate maycomprise a fusion polypeptide comprising the first immune co-stimulatorypolypeptide and the first member of the binding pair. In one specificembodiment, the first immune co-stimulatory polypeptide is selected fromthe group consisting of 4-1BBL, CD86, ICOSL, PD-L1, PD-L2, B7-H3, B7-H4,OX40L, CD27L, CD30L, LIGHT, BAFF, APRIL, CD80 and CD40L.

In one specific embodiment, the first antigen is associated with aninfectious agent, such as human or avian influenza or humanimmunodeficiency virus. In another specific embodiment, the firstantigen is a tumor associated antigen.

In one embodiment, the combination further comprises a third conjugatecomprising (i) a conjugate member comprising a second immuneco-stimulatory polypeptide and a first member of a binding pair and (ii)a conjugate member comprising a second antigen and a second member of abinding pair. In this embodiment, the second immune co-stimulatorypolypeptide is the same as or different from the first immuneco-stimulatory polypeptide; the second antigen is the same as ordifferent from the first antigen; the first and second binding pairmembers of the third conjugate are the same as or different from thefirst and second binding pair members of the first and secondconjugates. Additionally, the first conjugate member may be bound to thesecond conjugate member via binding between the first and second bindingpair members.

In another embodiment, the second conjugate of the combination comprises(i) a conjugate member comprising an infectious agent and (ii) aconjugate member comprising a second member of the binding pair.

In accordance with another aspect of the invention, there is provided amethod of generating or enhancing an immune response against a tumorwhich expresses a first tumor-associated antigen, comprisingadministering to a patient with the tumor (a) a first conjugatecomprising (i) a conjugate member comprising a first immuneco-stimulatory polypeptide and (ii) a conjugate member comprising afirst member of a binding pair, and a second conjugate comprising (i) aconjugate member comprising the first tumor-associated antigen and (ii)a conjugate member comprising a second member of the binding pair; or(b) immune cells which have been treated in vitro with the first andsecond conjugates.

In one embodiment, the first and second conjugates are administered tothe patient, separately or simultaneously, including as part of a singlecomposition.

In another embodiment, the patient is administered immune cells whichhave been treated in vitro with the first and second conjugates. In onespecific embodiment, the immune cells comprise a receptor for the immuneco-stimulatory polypeptide, and wherein the first conjugate isconjugated to the immune cells via binding between the immuneco-stimulatory polypeptide and the receptors, and the second conjugateis conjugated to the immune cell via binding between the first andsecond binding pair members.

In one embodiment, the method further comprises administering a thirdconjugate comprising (i) a conjugate member comprising a second immuneco-stimulatory polypeptide and a first member of a binding pair and (ii)a conjugate member comprising a second tumor-associated antigen and asecond member of a binding pair. In this embodiment, the second immuneco-stimulatory polypeptide is the same as or different from the firstimmune co-stimulatory polypeptide; the second antigen is the same as ordifferent from the first antigen; the first and second binding pairmembers of the third conjugate are the same as or different from thefirst and second binding pair members of the first and secondconjugates. Additionally, the first conjugate member may be bound to thesecond conjugate member via binding between the first and second bindingpair members.

In accordance with another aspect of the invention, there is provided amethod of modifying immune cells to generate or enhance an immuneresponse to a tumor expressing a tumor-associated antigen or to aninfectious agent, comprising contacting immune cells expressing areceptor for a first immune co-stimulatory polypeptide with (a) a firstconjugate comprising (i) a conjugate member comprising the first immuneco-stimulatory polypeptide and (ii) a conjugate member comprising afirst member of a binding pair; and (b) a second conjugate comprising(i) a conjugate member comprising an antigen associated with the tumoror infectious agent or the infectious agent and (ii) a conjugate membercomprising a second member of the binding pair. The first conjugate maybe conjugated to the immune cells via binding between the immuneco-stimulatory polypeptide and the receptor, and the second conjugatemay be conjugated to the immune cell via binding between the first andsecond binding pair members.

In one embodiment, the immune cell is a T cell, such as a CD4+ cell orCD8+ cell, or a neutrophil, natural killer cell, monocyte or dendriticcell.

In another embodiment, the immune cells comprise a receptor for a secondimmune co-stimulatory polypeptide and the method further comprisescontacting the immune cells with a third conjugate comprising (i) aconjugate member comprising the second immune co-stimulatory polypeptideand a first member of a binding pair and (ii) a conjugate membercomprising a second antigen associated with the tumor or infectiousagent or the infectious agent and a second member of the binding pair.In this embodiment, the second immune co-stimulatory polypeptide is thesame as or different from the first immune co-stimulatory polypeptide;the second antigen, if present, is the same as or different from thefirst antigen, if present; the first and second binding pair members ofthe third conjugate are the same as or different from the first andsecond binding pair members of the first and second conjugates.Additionally, the first conjugate member may be bound to the secondconjugate member via binding between the first and second binding pairmembers.

In accordance with another aspect of the invention, there is provided amodified immune cell expressing a receptor for a first immuneco-stimulatory polypeptide, wherein the modified immune cell is modifiedwith (a) a first conjugate comprising (i) a conjugate member comprisingthe first immune co-stimulatory polypeptide and (ii) a conjugate membercomprising a first member of a binding pair; and (b) a second conjugatecomprising (i) a conjugate member comprising a first antigen orinfectious agent and (ii) a conjugate member comprising a second memberof the binding pair, wherein the first conjugate is conjugated to theimmune cell via binding between the immune co-stimulatory polypeptideand the receptor, and the second conjugate is conjugated to the immunecell via binding between the first and second binding pair members. Inone embodiment, the immune cell is a T cell, such as a CD4+ cell or CD8+cell, or a neutrophil, natural killer cell, monocyte or dendritic cell.

In accordance with another aspect of the invention, there is provided amethod of inducing or enhancing an immune response against an infectiousagent, comprising administering to a patient suffering from or at riskof infection with the infectious agent (a) a first conjugate comprising(i) a conjugate member comprising a first immune co-stimulatorypolypeptide and (ii) a conjugate member comprising a first member of abinding pair; and (b) a second conjugate comprising (i) a conjugatemember comprising a first antigen associated with the infectious agentor comprising the infectious agent and (ii) a conjugate membercomprising a second member of the binding pair. In one embodiment, atleast one of the first and second conjugates is administered by directinjection into a site of infection.

In one specific embodiment, the infection is human or avian influenzaand the first antigen is selected from the group consisting of H, N, M1,M2e, NS1, NS2 (NEP), NP, PA, PB1, and PB2. In another specificembodiment, the infection is HIV and the first antigen is selected fromthe group of HIV antigens consisting of Gag proteins, Pol, Vif, Vpr,Rev, Vpu, envelope eptiopes, Tat, and Nef.

In one embodiment, the method further comprises administering a thirdconjugate comprising (i) a conjugate member comprising a second immuneco-stimulatory polypeptide and a first member of a binding pair and (ii)a conjugate member comprising a second antigen associated with theinfection or the infectious agent and a second member of the bindingpair, wherein the second immune co-stimulatory polypeptide is the sameas or different from the first immune co-stimulatory polypeptide; thesecond antigen, if present, is the same as or different from the firstantigen, if present; the first and second binding pair members of thethird conjugate are the same as or different from the first and secondbinding pair members of the first and second conjugates, and the firstconjugate member is bound to the second conjugate member via bindingbetween the first and second binding pair members.

In accordance with another aspect of the invention, there is provided aconjugate comprising an immune co-stimulatory polypeptide and avidin orstreptavidin.

In accordance with another aspect of the invention, there is provided amethod of inducing an immunostimulatory response in an animal comprisingadministering to the animal a conjugate comprising an immuneco-stimulatory polypeptide and avidin or streptavidin. In someembodiments, the method further comprises administering an antigen tothe animal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B set forth the nucleotide sequence (SEQ ID NO:1) andamino acid sequence (SEQ ID NO:2), respectively, of a fusion proteincomprising core streptavidin and the extracellular domain of the murineLIGHT protein. The core streptavidin sequence is underlined in FIG. 1B.

FIGS. 2A and 2B set forth the nucleotide sequence (SEQ ID NO:3) andamino acid sequence (SEQ ID NO:4), respectively, of a fusion proteincomprising the extracellular domain of human CD80 and core streptavidin.The core streptavidin sequence is underlined in FIG. 2B.

FIGS. 3A and 3B set forth the nucleotide sequence (SEQ ID NO:5) andamino acid sequence (SEQ ID NO:6), respectively, of a fusion proteincomprising the extracellular domain of murine 4-1BBL and corestreptavidin. The core streptavidin sequence is underlined in FIG. 3B.

FIGS. 4A and 4B set forth the nucleotide sequence (SEQ ID NO:7) andamino acid sequence (SEQ ID NO:8), respectively, of a fusion proteincomprising core streptavidin and the extracellular domain of human4-1BBL. The core streptavidin sequence is underlined in FIG. 4B.

FIGS. 5A and 5B set forth the nucleotide sequence (SEQ ID NO:9) andamino acid sequence (SEQ ID NO:10), respectively, of a fusion proteincomprising core streptavidin and the extracellular domain of human CD86.The core streptavidin sequence is underlined in FIG. 5B.

FIGS. 6A, 6B and 6C set forth the amino acid sequences of HPV16 E6 (SEQID NO:11), an HPV16 E6 variant (SEQ ID NO:12), and HV16 E7 (SEQ IDNO:13).

FIGS. 7A & 7B set forth the nucleotide and amio acid sequences of theCSA-human CD40L (SEQ ID NOs 14 & 15) constructs used in the examples.

FIG. 8 shows the results of allogenic mixed lymphocyte reactions usingnaïve BALB/c lymphocytes as responders and allogeneic C57BL/6 irradiatedsplenocytes as stimulators. Indicated cultures were supplemented with 1μg/ml CSA-41BBL fusion protein.

FIG. 9 shows the results of ex vivo T cell proliferation where CD8⁺ Tcells sorted from C57B/6 mice were stimulated with soluble anti-CD3monoclonal antibody (0.5 μg/ml) and irradiated splenocytes in thepresence or absence of CSA-41BBL fusion protein (0.5 μg/ml), control CSAprotein (0.19 μg/ml), or anti-4-1BB monoclonal antibody clone 3H3 (5μg/ml).

FIG. 10 shows the proliferative response of antigen specific CD8+ Tcells when one million OT-I CD8⁺ T cells were labeled with CFSE andtransferred into B6.SJL mice that were immunized with biotinylated OVA(10 μg/injection) and CSA-4-1BBL fusion protein (1 μg/injection) mixedwith biotinylated OVA (41BBL+OVA) or a conjugate comprising biotinylatedOVA and CSA-4-1BBL. The last panel (*) shows the response for 5 μg ofCSA-4-1BBL conjugated to 10 μg biotinylated OVA. Core streptavidin (SA)was used at equimolar level as 4-1BBL.

FIG. 11A is a histogram showing PE+ cells of untreated DC (gray filledarea), DC treated with biotinylated PE (dashed line) and DC treated withbiotinylated PE/CSA-4-1BBL conjugate (solid line). FIG. 11B shows themean fluorescence intensity (MFI) of PE for DC receiving each treatment.

FIG. 12A shows the results of flow cytometry performed to analyze CD86and MHC class II levels of DC untreated (dark gray) or treated withCSA-41BBL (solid line) or LPS (dashed line) in the presence of GM-CSF.FIG. 12B shows the mean fluorescence intensity of CD86 and MHC class II.

FIG. 13 shows the mean florescence intensity of CD40, CD86 and MHC classII expression on DC cells from naïve, biotinylated OVA/CSA treated, andbiotinylated OVA/CSA-4-1BBL treated animals.

FIG. 14A shows the results of coculture experiments where CD4⁺ CD25⁻(single positive, SP) and CD4⁺ CD25⁺ (double positive, DP) T cells weresorted from the spleen and peripheral lymph nodes of naïve BALB/c miceand cultured alone or at 1:1 ratio for 3 days, in cultures supplementedwith irradiated splenocytes, an anti-CD3 antibody (0.5 μg/ml), andindicated concentrations (μg/ml) of 4-1BBL or equimolar amount ofcontrol CSA protein. FIG. 14B shows the results of a CFSE assay where SPT cells labeled with CFSE were used in a suppression assay underconditions described for FIG. 14A, except that 4-1BBL was used at 0.5μg/ml. The percentage of dividing cells is shown for each histogram.

FIG. 15 shows the results of ex vivo T cell proliferation where CD4⁺CD25⁻ (single positive, SP) and CD4⁺CD25⁺ (double positive, DP) T cellswere sorted from the spleen and peripheral lymph nodes of naïve BALB/cmice, and cultured alone or in a 1:1 ratio in the presence of 0.5 μg/mlanti-CD3 antibody and irradiated splenocytes with or without 1 μg/ml of4-1 BBL.

FIG. 16 shows the construction of CSA-hCD40L and CSA-mCD40L constructs.Arrows indicate primers (a, b, c, d) and their orientations used forcloning purposes.

FIG. 17 shows the flow cytometry analysis demonstrating binding ofCSA-mCD40L and CSA-hCD40L to CD40 receptors, where human THP-1 and mouseA20 cell lines were incubated with CSA-mCD40L or CSA-hCD40L, stainedwith FITC labeled anti-streptavidin antibody, and analyzed in flowcytometry. Panels (a) and (b) show binding of CSA-mCD40L to human andmouse cell lines, respectively, and panels (c) and (d) show binding ofCSA-hCD40L to human and mouse cell lines, respectively.

FIG. 18 shows the flow cytometry analysis demonstrating upregulation ofHLA class II and costimulatory molecules on macrophages stimulated withCSA-hCD40L, where a human THP-1 cell line was stimulated with 100 ng/mlof CSA-hCD40L for 48 hours (thin solid lines) and analyzed usingantibodies to HLA class II (FIG. 18A) and CD80 (FIG. 18B) molecules inflow cytometry. Cells incubated with CSA protein (solid histograms) andCHO cell transfectants expressing membrane-bound mouse CD40L (thicksolid line) served as negative and positive controls, respectively.

FIG. 19 shows the flow cytometry analysis demonstrating the phenotypicmaturation of murine DCs stimulated with CSA-mCD40L, where bonemarrow-derived immature DCs were stimulated with varying concentrationsof CSA-mCD40L (open histograms) for various time periods, and analyzedusing specific antibodies for the expression of CD80 (FIG. 19A) and CD86(FIG. 19B). Cells left unstimulated (thin solid lines) or stimulatedwith CSA protein (solid histograms) served as controls. Data are shownfor 200 ng protein per 10⁶ cells stimulated for 48 hours.

FIG. 20 shows the secretion of cytokines by human monocytes stimulatedwith CSA-CD40L, where primary elutriated human monocytes were stimulatedwith 1 μg/ml of each rhsCD40L+ enhancer (FIG. 20A) and 100 ng/ml ofCSA-hCD40L (FIG. 20B) or 250 ng/ml of CSA-mCD40L (FIG. 20C) or CSA for18 hours, and supernatants were analyzed for IL-1β and IL-6 content byELISA. FIG. 20D shows the results when a murine macrophage cell linegenetically modified to express human CD40 (CD40KO) was stimulated withCSA or CSA-hCD40L (1 μg/ml) for 3 hours and RNA was extracted andanalyzed by RNAse protection assay using the RiboQuant multiprobe RNAseprotection system with the template mck-3b. The protected probes forIL-6, L32, and GAPDH are shown. The histogram represents the banddensity of IL-6 after normalization with the housekeeping gene L32.

FIG. 21 shows the stimulation of iNOS expression in macrophagesstimulated with CSA-hCD40L, where the murine CD40KO macrophage linetransfected to express human CD40 was primed with IFN-γ for 24 hours andcells were then stimulated with 1 μg/ml of commercial rhsCD40L with theenhancer or 300 ng/ml of CSA-hCD40L or CSA proteins for 24 hours, andcell lysates were analyzed by Western blot using anti-iNOS antibody. Thehistogram represents the density of the iNOS bands.

FIG. 22 shows the strong in vivo adjuvant effect of CSA-4-1BBL ascompared to LPS at doses of 12.5 and 25 μg, with 50 μg OVA as theantigen. Results are reported in terms of in vivo killing percentage.

FIG. 23 shows the effects on existing cervical tumors of vaccinationwith (i) PBS (♦, n=20); (ii) 50 μg P1+12.5 μg CSA (, n=6) (iii) 25 μgCSA-4-1BBL (▴, n=10); (iv) 50 μg P1+25 μg CSA-4-1BBL (Δ, n=13), or (v)50 μg P1+10 μg CpG (□, n=7). Vaccination with the combination of P1 andCSA-4-1BBL resulted in significantly enhanced survival rates, whilevaccination with either P1 or CSA-4-1BBL provided some successfulimmunotherapy.

FIG. 24 shows the results of vaccination with a biotinylatedOVA/CSA-4-1BBL conjugate in preventing tumor growth. Tumor-free survivalof mice vaccinated with OVA (Δ), a biotinylated OVA/CSA-4-1BBL conjugate(▴) and control mice () are shown, with mice vaccinated with thebiotinylated OVA/CSA-4-1BBL conjugate showing 100% survival.

FIG. 25 shows flow cytometry of CFSE stained cells, demonstrating that4-1BBL could enhance the antigen specific CTL response in vivo to higherlevels compared to antigen alone, or antigen and LPS. Results areexpressed on the corner of each panel as percentage lysis of the peptidepulsed CFSEhi peak as compared with the reference CFSElow peaknormalized to naïve animal.

FIG. 26 shows flow cytometry data demonstrating that 4-1BBLcostimulation increased antigen presentation in vivo.

FIG. 27 shows flow cytometry data demonstrating that 4-1BBLcostimulation increases antigen uptake by dendritic cells in vivo.

DETAILED DESCRIPTION

The present invention provides methods and compositions for generatingor enhancing an immune response, including an immune response against anantigen, such as a TAA or antigen associated with an infectious agentsuch as human and avian influenza or HIV. The invention also providesmodified immune cells that are useful for generating or enhancing animmune response to an antigen. The invention also provides immunotherapymethods, including cancer immunotherapy methods, such as methods ofreducing tumor size and methods of inhibiting the growth of tumor cells,and methods of treating or preventing infections.

A productive adaptive immune response requires coordinated and timelyinteractions between naïve T effector (“Teff”) cells and APCs within theorganized structures of secondary lymphoid organs. This interactionpromotes reciprocal activation of Teff cells and APCs, leading to theexpression of various cell surface ligands and receptors as well assoluble proteins that are important for the initiation, maintenance, andlong-term memory of the response. As discussed above, at least threesignals (Signal 1, 2, and 3), are involved in the initial activation ofnaïve T cells. Several immune co-stimulatory molecules have beenimplicated that stimulate one or more of these Signals.

The present invention relates to the use of one or more immuneco-stimulatory polypeptides and one or more antigens associated with atumor or infectious agent in methods that present the antigen to immunecells such that an effective immune response against the tumor orinfectious agent is induced. Alternatively, an infectious agent can beused in place of an antigen associated therewith. While not wanting tobe bound by any theory, it is believed that the present inventionachieves advantageous results by facilitating antigen presentation andactivating the immune response. In another alternative embodiment, theinvention provides immunostimulatory moieties comprising an immuneco-stimulatory polypeptide, that are useful for stimulating an immuneresponse.

For the purposes of the present application, the following terms havethese definitions:

As used herein “a” or “an” means one or more, unless specificallyindicated to mean only one.

“Administration” as used herein encompasses all suitable means ofproviding a substance to a patient. Common routes include oral,sublingual, transmucosal, transdermal, rectal, vaginal, subcutaneous,intramuscular, intravenous, intra-arterial, intrathecal, via catheter,via implant etc. In some embodiments, a composition is administered nearor directly to the tumor, such as by direct injection into the tumor orinjection into the blood such as when the tumor is a tumor of the blood.

“Antigen” is used herein without limitation. Antigens include proteins,lipids, sugars, nucleic acids, chemical moieties, and other moietiesthat induce an immune response. Antigens include proteins, which may ormay not be modified, such as by glycosylation or methylation, that arecyclized or bound to lipids, for example. Antigens associated with aninfectious agent or disease include antigens that are part of theinfectious agent, such as envelope proteins, capsid proteins, surfaceproteins, toxins, cell walls, antigenic lipids, and the like. Otherantigens may be expressed only in the presence of the host. Othersuitable antigens may, in some embodiments, include antigens of thehost, including those that are induced, modified or otherwiseoverexpressed as a marker of infection or disease. All such antigensthat are derived from, or associated with, an infectious agent, aninfection, a condition or disease, are suitable for use in the presentinvention. Also suitable for use as an “antigen” in accordance with thepresent invention are peptides comprising antigenic portions offull-length proteins, such as peptides comprising a portion of a proteinthat induces an immune response, such as an immunogenic epitope. Forexample, suitable antigens may include synthetic peptides that induce animmune response.

“Binding pair” refers to two molecules which interact with each otherthrough any of a variety of molecular forces including, for example,ionic, covalent, hydrophobic, van der Waals, and hydrogen bonding, sothat the pair have the property of binding specifically to each other.Specific binding means that the binding pair members exhibit binding toeach other under conditions where they do not bind to another molecule.Examples of binding pairs are biotin-avidin, hormone-receptor,receptor-ligand, enzyme-substrate, IgG-protein A, antigen-antibody, andthe like.

“Immune co-stimulatory polypeptide” means a polypeptide that increasesan individual's immune response against a pathogen (including aninfectious agent) or tumor.

“Immune cell” as used herein includes any cell that is involved in thegeneration, regulation or effect of the acquired or innate immunesystem. Immune cells include T cells such as CD4+ cells, CD8+ cells andvarious other T cell subsets, B cells, natural killer cells,macrophages, monocytes and dendritic cells, and neutrophils.

“Patient” as used herein includes any vertebrate animal, includingequine, ovine, caprine, bovine, porcine, avian, canine, feline andprimate species. In one embodiment, the patient is human. A person ofordinary skill in the art will recognize that particular immuneco-stimulatory molecules, signaling molecules, cell markers, cell types,infectious agents etc., discussed with reference to one species, mayhave corresponding analogues in different species, and that suchanalogues, and their use in corresponding and related species, areencompassed by the present invention.

“Tumor” as used herein includes solid and non solid tumors (such asleukemia); and different stages of tumor development from pre-cancerouslesions and benign tumors, to cancerous, malignant and metastatictumors.

In general terms, the invention provides methods whereby an immuneresponse against a first antigen is generated or enhanced using (a) afirst conjugate comprising (i) a conjugate member comprising a firstimmune co-stimulatory polypeptide and (ii) a conjugate member comprisinga first member of a binding pair, and (b) a second conjugate comprising(i) a conjugate member comprising the first antigen and (ii) a conjugatemember comprising a second member of the binding pair. The antigen maybe a TAA or an antigen associated with an infectious agent, or theinfectious agent itself. The conjugates may be administered directly toa patient comprising the antigen or infectious agent, or may be used totreat immune cells which then are administered to the patient. Theinvention also provides compositions comprising the conjugates, immunecells treated with the conjugates, and methods of making treated immunecells.

The invention also provides immunostimulatory moieties comprising animmune co-stimulatory polypeptide, such as a conjugate or fusion proteincomprising an immune co-stimulatory polypeptide and avidin orstreptavidin. The invention also provides a method of inducing animmunostimulatory response in an animal comprising administering animmunostimulatory moiety to the animal. In some embodiments, an antigenalso is administered to the animal. Compositions comprising the moietyalso are provided.

In accordance with one aspect of the invention, the antigen orinfectious agent is presented to immune cells as part of a conjugatecomprising an immune co-stimulatory polypeptide that selectively targetsone or more types of immune cells, such as any of the immuneco-stimulatory polypeptides described below. Thus, in accordance withone embodiment, the invention provides a conjugate comprising an immuneco-stimulatory polypeptide and an antigen associated with a tumor orinfectious agent or the infectious agent. The antigen or infectiousagent can be conjugated to the immune co-stimulatory polypeptide by anymeans, including by covalent bonding, directly or through a linker, orthrough binding pair members.

In one embodiment, the antigen or infectious agent is conjugated to theimmune co-stimulatory polypeptide through the binding interactions of abinding pair. In accordance with this embodiment, each of the antigen(or infectious agent) and the immune co-stimulatory polypeptide isconjugated to a member of a binding pair, and the binding interactionsof the binding pair members link the antigen (or infectious agent) andimmune co-stimulatory polypeptide together in a conjugate, such as animmune co-stimulatory polypeptide-first binding pair member::secondbinding pair member-antigen (or infectious agent) conjugate.

In accordance with this embodiment, the invention provides a combinationcomprising (a) a first conjugate comprising (i) a conjugate membercomprising a first immune co-stimulatory polypeptide and (ii) aconjugate member comprising a first member of a binding pair, and (b) asecond conjugate comprising (i) a conjugate member comprising a firstantigen associated with a tumor or infectious agent (or the infectiousagent itself) and (ii) a conjugate member comprising a second member ofthe binding pair.

In another embodiment, the combination comprises a third conjugatecomprising (i) a conjugate member comprising a second immuneco-stimulatory polypeptide and (ii) a conjugate member comprising asecond antigen associated with the tumor or infectious agent (or theinfectious agent itself), wherein the immune co-stimulatory polypeptideof the third conjugate is the same as or different from the immuneco-stimulatory polypeptide of the first conjugate and the second antigenis the same as or different from the first antigen. In a specific aspectof this embodiment, the immune co-stimulatory polypeptide and secondantigen of the third conjugate are bound together via binding betweenbinding pair members associated with each of the immune co-stimulatorypolypeptide and second antigen. In accordance with this embodiment, thebinding pair members of the third conjugate may be the same as ordifferent from the first and second biding pair members of the first andsecond conjugates.

The first, second and optional third conjugates may be provided inseparate compositions. Alternatively, the first and second conjugatesmay be provided in a single composition, and the third conjugate may beprovided in a separate composition. In yet another alternative, thefirst, second and third conjugates are provided in a single composition.In another alternative, the first conjugate is provided in onecomposition and the second and third conjugates are provided in anothercomposition

Each composition optionally may comprise a pharmaceutically acceptablecarrier. A pharmaceutically acceptable carrier is a material that can beused as a vehicle for the composition because the material is inert orotherwise medically acceptable, as well as compatible with the activeagent(s), in the context of administration. A pharmaceuticallyacceptable carrier can contain conventional pharmaceutical additives asare well known in the art.

Immune Co-Stimulatory Polypeptides

Immune co-stimulatory molecules are involved in the natural interactionbetween naïve T cells and antigen presenting cells, which results intheir reciprocal activation and prompts the expression of various cellsurface ligands and receptors, and soluble proteins that contribute tothe initiation, maintenance, and long-term memory of the immuneresponse. As discussed above, at least three signals are required forthe initial activation of naïve T cells. Signal 1 is generated byinteractions between a T cell receptor (TCR) and a nominal peptidepresented by major histocompatibility complex (MHC) molecules on thesurface of professional APC, such as dendritic cells (DC). Signal 2 canbe mediated by several different molecules and is important to asustained immune response. Signal 3 is transduced via cytokineselaborated by activated T cells and APC and is important to themaintenance of effector immune responses.

A number of immune co-stimulatory molecules have been identified.Exemplary immune co-stimulatory molecules (polypeptides) useful inaccordance with the invention include, without limitation, LIGHT, CD80(B7-1), CD86 (B7-2), ICOS, ICOSL (including B7h, B7-H2, B7RP-1, GL-50and LICOS), CD94 (KP43), CD40L (CD154), ICAM-1 (CD54), ICAM-2, ICAM-3,SLAM (CD150), HAS (CD24), 4-1BB (CDw137), 4-1BBL (CDw137L), OX40L, CD28,CD40 (BP50), CD25 (IL-2R α), Lymphotoxin (LTα or LTβ), TNF, Fas-L, GITR(activation-inducible TNRF), GITR Ligand, CD11a (α_(L) integrin), CD11b(α_(M) integrin), L-selectin (CD62L), CD69 (very early activationantigen), CD70 (CD27L), PD-L1, PD-L2, B7-H3, B7-H4, OX40L, 4-1BBL,CD27L, CD30L, LIGHT, BAFF, and APRIL. See, e.g., Watts & DeBenedette,1999, Curr. Opin. Immunol., 11:286-93.

Unless specified herein as “full-length,” reference herein to an immuneco-stimulatory polypeptide encompasses the full-length polypeptide aswell as fragments or portions thereof that exhibit immune co-stimulatoryfunction, including, but not limited to those fragments and portionsspecifically identified below. Thus, for example, reference to a 4-1BBLpolypeptide connotes a polypeptide comprising a fragment or portion offull-length 4-1 BBL that exhibits immune co-stimulatory function, suchas the extracellular domain of 4-1BBL or the full-length 4-1BBL protein.In one embodiment, the immune co-stimulatory polypeptide does notcomprise the transmembrane domain of an immune co-stimulatory molecule.In one embodiment, the immune co-stimulatory polypeptide comprises theextracellular domain of an immune co-stimulatory molecule, or a receptorbinding fragment thereof.

Examples of representative nucleic acid sequences and the encoded immuneco-stimulatory polypeptides include those shown in GenBank AccessionNos. AB029155 (murine LIGHT); NM_(—)172014 (human TNFSF14 mRNAtranscript variant 2); NM_(—)003807 (human TNFSF14 mRNA transcriptvariant 1); NM_(—)005191 (human CD80 mRNA); NM_(—)009855 (mouse CD80mRNA); NM_(—)214087 Sus scrofa CD80 mRNA); NM_(—)009404 (murine Tnfsf9mRNA); NM_(—)003811 (human TNFSF9 mRNA); NM_(—)181384 (Rattus norvegicusTnfsf9 mRNA); BAA88559 (murine LIGHT protein); Q9QYH9 (murine TNFSF14membrane bound protein and soluble protein); AAH18058 (human TNFSF14protein); NP_(—)005182 (human CD80 protein); NP_(—)033985 (murine CD80protein); NP_(—)037058 (Rattus norvegicus CD80 protein); NP_(—)003802(human TNFSF9 protein); NP_(—)033430 (mouse TNFSF9 protein);NP_(—)852049 (Rattus norvegicus TNFSF9 protein); NM_(—)012967 (Rattusnorvegicus ICAM-1 mRNA); X69711 (human ICAM-1 mRNA); X52264 (murineICAM-1 mRNA); X69819 (human ICAM-3 mRNA); AF296283 (murine ICAM-4 mRNA);NM_(—)021181 (human SLAMF7 mRNA); NM_(—)033438 (human SLAMF9 mRNA);NM_(—)029612 (murine SLAMF9 mRNA); NM_(—)144539 (murine SLAMF7 mRNA);L13944 (murine CD18 gene); X53586 (human integrin α6 mRNA); X68742(human integrin α mRNA); J04145 (Human neutrophil adherence receptoralpha-M subunit mRNA); AJ246000 (human leucocyte adhesion receptor,L-selectin mRNA); AY367061 (human L-selectin mRNA, partial cds); Y13636(murine CD70 mRNA); NM_(—)001252 (human TNFSF7 mRNA); BC000725 (humanTNFSF7 mRNA (cDNA clone MGC:1597 IMAGE:3506629), complete cds); X69397(human CD24 gene and complete CDS); NM_(—)013230 (human CD24 mRNA);NM_(—)012752 (Rattus norvegicus CD24 mRNA); Y00137 (murine tumornecrosis factor-beta (lymphotoxin) gene); X02911 (human tumor necrosisfactor-beta (lymphotoxin) gene); D00102 (human lymphotoxin mRNA,complete CDS); X01393 (human lymphotoxin mRNA); and A06316 ((humanlymphotoxin mRNA). Other nucleic acid sequences encoding the same orother immune co-stimulatory polypeptides and/or amino acid sequences ofco-stimulatory polypeptides can be found, for example, by searching thepublicly available GenBank database (available, for example, atncbi.nlm.nih.gov on the World Wide Web).

Interactions between CD28 and CD80/CD86 appear to play a significantrole in the transduction of Signal 2. See, e.g., Harding & Allison, J.Exp. Med. 1993, 177: 1791-96; Ramarathinam et al., J. Exp. Med. 1994,179: 1205-14; Townsend & Allison, Science 1993, 259: 368-70; Gause etal., J. Immunol. 1997, 159: 1055-58. CD80 is usually not expressed onresting B cells and is expressed at low levels on peripheral bloodmonocytes and DC; however, both of these cells as well as macrophagesand other APCs upregulate their expression of CD80 following activation.See, e.g., Lenschow et al., Ann. Rev. Immunol. 1996, 14: 233-58; Freemanet al., J. Immunol. 1989, 143:2714-22. In contrast, CD86 isconstitutively expressed on peripheral blood monocytes and DC and morerapidly upregulated on B cells. See, e.g., Lenschow et al., supra, Inabaet al., J. Exp. Med. 1994, 180: 1849-60. TCR interaction withMHC/peptide complex on APCs allows for simultaneous engagement ofCD80/86 molecules with CD28 and leads to the tyrosine phosphorylation ofthe lipid kinase phosphatidylinositol 3-kinase, which in turn initiatesa series of complex intracellular events that result in the induction ofIL-2 gene expression, cell proliferation, and differentiation intoeffector function. See, e.g., Slavik et al., Immunol. Res. 1999, 19:1-24; Azuma et al., Nature 1993, 366: 76-79; Allison & Krummel, Science1995, 270: 932-33.

Signal 2 may further augment a productive immune response by preventingcell death through the regulation of anti-apoptotic genes, such asBcl-xL. See, e.g., Radvanyi et al., J. Immunol. 1996, 156: 1788-98;Boise et al., Immunity 1995, 3: 87-98; Boise & Thompson, Science 1996,274: 67-68. Following the initial stages of immune activation, a numberof additional receptor-ligand pairs are upregulated on the surface of Tcells and APCs. These “secondary” receptor/ligand pairs, such as4-1BBL/4-1BB, play important roles in the maintenance of post initialactivation events, immune homeostasis, and generation of immunologicalmemory. See, e.g., Yu et al., Nat. Immunol. 2004, 5: 141-49; Armitage etal., Nature 1992, 357: 80-82; Zhai et al., J. Clin. Invest. 1998, 102:1142-51; Bourgeois et al. Science 2002, 297: 2060-63; Kikuchi et al.,Nat. Med. 2000, 6: 1154-59.

1. 4-1BBL

In one particular embodiment of the invention, the immune co-stimulatorypolypeptide is a 4-1BBL polypeptide. 4-1BBL (also known as 4-BB-L, 4-BBligand, TNFSF9, ILA ligand) is a type II protein expressed on activatedB cells, macrophages, and DC two to three days following activation.See, e.g., Alderson et al. Eur. J. Immunol. 1994, 24: 2219-27; Goodwinet al., Eur. J. Immunol. 1993, 23: 2631-41; Pollok et al., Eur. J.Immunol. 1994, 24: 367-74; DeBenedette et al., J. Immunol. 1997, 158:551-59. Its receptor, 4-1BB (CD137), is expressed on the surface ofactivated CD4⁺ and CD8⁺ T cells, on natural killer cells, monocytes, andresting DC. See, e.g., Pollock, supra; Wilcox et al., J. Immunol. 2002,169: 4230-36; Futagawa et al., Int. Immunol. 2002, 14: 275-86; Pollok etal., J. Immunol. 1993, 150: 771-81.

4-1BB/4-1BBL interactions also transduce Signal 2 to CD8⁺ T cells in aCD28-independent manner and stimulate them to produce cytokines, expand,and acquire effector functions. See, e.g., Cannons et al., J. Immunol.2001, 167: 1313-24; Hurtado et al., J. Immunol. 1995, 155: 3360-67. Kim& Broxmeyer, J. Hematother. Stem Cell Res. 2001, 10: 441-49; Saoulli etal., J. Exp. Med. 1998, 187: 1849-62; Shuford et al., J. Exp. Med. 1997,186:47-55; Tan et al., J. Immunol. 1999, 163: 4859-68; Vinay & Kwon,Semin. Immunol. 1998, 10: 481-89. 4-1BB/4-1BBL interaction is alsoimportant for the activation of monocytes and DC, their synthesis ofcytokines, and communication with NK cells. See, e.g., Futagawa et al.,supra; Wilcox et al., J. Clin. Invest 2002, 109: 651-9. Similarly, inaddition to its role in promoting the expansion of antigen-specific Tcells through the upregulation of cyclins D2 and E, and downregulationof cyclin-dependent kinase inhibitor p27kip1, 4-1BB signaling plays arole in T cell survival, as it prevents activation-induced cell deathvia the upregulation of the anti-apoptotic Bcl-xL and Bcl-2 and theestablishment of long-term immunological memory. See, e.g., Takahashi etal., J. Immunol. 1999, 162: 5037-40; Hurtado et al., J. Immunol. 1997,158: 2600-09; Kim et al., Eur. J. Immunol. 1998, 28: 881-90.4-1BB/4-1BBL interaction has also been shown to selectively promote type1 cytokines, such as IL-2, IFN-γ, and TNF-α, suggesting that 4-1BB maybe a costimulatory molecule specific for type 1 effector T cells, whichplay a role in tumor eradication.

It has recently been shown that T_(reg) cells constitutively express4-1BB receptor and that signal transduction through 4-1BB receptorinhibits the suppressive function of these cells. See, e.g., Choi etal., supra; Morris et al., supra, and the Examples below. This isimportant because T_(reg) cells play a significant role in tumor evasionof the immune system. Several clinical studies demonstrated that adirect correlation exists between the number of T_(reg) cells and tumorprogression. Curiel et al., Nat. Med. 2004, 10: 942-49. Indeed, theeradication of T_(reg) cells in animal models has resulted in theeradication of large tumors, providing direct evidence for theirdominant role in tumor progression. Yu et al, J. Exp. Med. 2005, 201:779-91. Similarly, infectious agents, such as HIV, may use T_(reg) cellsfor immue evasion.

Although not wishing to be bound by theory, it is believed that the useof 4-1BBL as an immune co-stimulatory polypeptide in accordance with theinvention may activate the 4-1BB cognate receptor on T cells, resultingin several important immune-stimulatory effects. One effect may be thetransduction of Signal 2 to CD8⁺ T cells in a CD28-independent manner,which stimulates the T cells to produce cytokines, to expand, andacquire effector functions. Another effect of 4-1BB/4-1BBL interactionmay be activation of monocytes and DC which results in the synthesis andrelease of cytokines. Yet another effect of 4-1BB signaling may be thepromotion of T cell survival and establishment of long-termimmunological memory by preventing activation-induced cell death (AICD).Still another effect of 4-1BB/4-1BBL interaction may be the selectiveproduction of type 1 cytokines, such as IL-2, IL-12, IFN-γ, and TNF-αfrom T cells, DC and macrophages, which act upon type 1 effector T cellsimportant to tumor eradication. Also, as explained above, 4-1BB/4-1BBLinteraction may inhibit the suppressor function of T_(reg) cells. Thus,for example, a 4-1BBL-antigen conjugate may specifically bind to DCexpressing the 4-1BB receptor, facilitate antigen presentation, activateDC for the generation of a primary T cell response, directly act onactivated T cells (including T_(eff) cells) and NK cells to boost theirresponse against the antigen, and inhibit the suppressive function ofT_(reg) cells.

4-1BBL contains 254 amino acids (26624 Da). See Alderson et al. Eur J.Immunol. 1994 September; 24(9):2219-27. The full amino acid sequence ofhuman 4-1BBL can be found under accession no. P41273 in the Swiss-Protdatabase. 4-1BBL is a type II glycoprotein with residues 1-28 forming apotential cytoplasmic domain, residues 29-49 forming a single predictedtransmembrane domain, residues 50-254 forming a potential extraceulluardomain, and residues 35-41 representing a poly-Leu stretch. Thenucleotide sequence in humans encoding the 4-1BBL can be found inGenBank accession no. NM_(—)003811.

As discussed above, 4-1BBL is expressed by activated antigen presentingcells including activated B cells, macrophages, and DC, 2-3 daysfollowing activation. 4-1BB, which is the receptor for 4-1BBL, isexpressed on the surface of activated CD4⁺ and CD8⁺ T cells, on naturalkiller cells, monocytes, and resting DC. Residues 50-254 of 4-1BBL orfragments thereof that can bind to its cognate receptor 4-1BB, can belinked or expressed as a fusion with a binding pair member for use inaccordance with the present invention. For example, FIGS. 3A and B showthe nucleotide and amino acid sequences of a CSA-murine 4-1BBL fusionprotein (SEQ ID NOs 5 and 6). FIGS. 4A and B show the nucleotide andamino acid sequences of a fusion protein comprising the extracellulardomain of human 4-1BBL and core strepavidin (SEQ ID NOs 7 and 8).

2. CD80 & CD86

CD80 (also known as B7.1, CD28LG, LAB7) and CD86 (also known as B7.2,CD28LG2, LAB72) are exemplary co-stimulatory polypeptides, both of whichbind to the CD28/CTLA4 co-receptor expressed by T cells. CD80 contains288 amino acids (33048 Da). See Freeman et al. J. Immunol. 143 (8),2714-2722 (1989). The full amino acid sequence of human CD80 can befound under accession no. P33681 in the Swiss-Prot database. CD80 is atype I glycoprotein with residues 1-34 forming a secretion signal,residues 35-242 forming a potential extraceulluar domain, residues243-263 forming a potential transmembrane domain, and residues 264-288forming a potential cytoplasmic domain. Thus the mature CD80 moleculewithout its secretion signal sequence represents amino acids 35-288. Thenucleotide sequence in humans encoding CD80 can be found in GenBankaccession no. NM_(—)005191.

Residues 35-242 of CD80 or fragments thereof that can bind to itscognate receptor CD28 can be linked or expressed as a fusion proteinwith a binding pair member for use in accordance with the presentinvention. For example, FIGS. 2A and 2B set forth the nucleotides (SEQID NO:3) and amino acid sequence (SEQ ID NO:4) of a fusion proteincomprising the extracellular domain of human CD80 (B7.1) and corestreptavidin.

CD86 (B7.2) contains 329 amino acids (37696 Da). See Freeman et al.Science 262 (5135), 909-911 (1993). The full amino acid sequence ofhuman CD86 can be found under accession no. P42081 in the Swiss-Protdatabase. CD86 is a type I glycoprotein with residues 1-23 forming asecretion signal, residues 24-247 forming a potential extraceulluardomain, residues 248-268 forming a potential transmembrane domain, andresidues 269-329 forming a potential cytoplasmic domain. Thus, themature CD86 molecule without its secretion signal sequence representsamino acids 24-329. The nucleotide sequence in humans encoding CD86 canbe found in GenBank accession no. NM_(—)175862.

Residues 24-247 of CD86 or fragments thereof that can bind to itscognate receptor CD28, can be linked or expressed as a fusion with abinding pair member for use in accordance with the present invention.For example, FIGS. 5A and 5B set forth the nucleotide (SEQ ID NO:9) andamino acid (SEQ ID NO: 10) sequences of a fusion protein comprising theextracellular domain of human CD86 (B7.2) and core streptavidin.

CD86 is usually not expressed on resting B cells and is expressed at lowlevels on peripheral blood monocytes (PBC) and DC. Its expression,however, is upregulated on B cells and other APC such as macrophages andDC following activation. In contrast, CD86 is constitutively expressedon PBC and DC and more rapidly upregulated on B cells. T cell receptor(TCR) interaction with the MHC/peptide complex on APC allows forsimultaneous engagement of CD80/86 with CD28 on the T cell, which leadsto tyrosine phosphorylation of the lipid kinase phosphotidylinositol3-kinase, which in turn initiates a series of intracellular events thatresult in the induction of IL-2 gene expression, cell proliferation, anddifferentiation into effector function. Signal 2 may further augment aproductive immune response by preventing cell death through theregulation of antiapoptotic genes, such as Bcl-xL.

3. LIGHT

Following the initial stages of immune activation, “secondary”receptor/ligand pairs such as 4-1BBL/4-1BB (discussed above) andLIGHT/HVEM become upregulated on the surface of T cells and APC. Thesereceptor/ligand pairs are involved in the maintenance of post initialactivation events, immune homeostasis, and generation of immunologicalmemory.

The LIGHT polypeptide (also known as TNFS14, HVEM-L, LTg, TR2) is a TNFsuperfamily member which is homologous to lymphotoxin. See Mauri et al.Immunity 8 (1), 21-30 (1998). The full amino acid sequence of humanLIGHT can be found under accession no. O43557 in the Swiss-Protdatabase. LIGHT contains 240 amino acids (26351 Da) and is a type IIglycoprotein with residues 1-37 forming a potential cytoplasmic domain,residues 38-58 forming a single predicted transmembrane domain, andresidues 59-240 forming a potential extraceulluar domain. A cleavagesite involves residues 82-83. The nucleotide sequence in humans encodingLIGHT can be found in GenBank accession no. NM_(—)172014.

Residues 59-240 of LIGHT or fragments thereof that can bind to itscognate receptor HVEM, LTβR or TR6, can be linked or expressed as afusion with a binding pair member for use in accordance with the presentinvention. For example, FIGS. 1A and 1B set forth the nucleotide (SEQ IDNO:1) and amino acid sequences (SEQ ID NO:2) of a fusion comprising corestreptavidin and the extracellular domain of murine LIGHT.

LIGHT is primarily expressed on activated T cells, NK cells, andimmature dendritic cells, and serves to regulate various aspects ofimmune responses. LIGHT is synthesized as a membrane-bound protein, butits cell-surface expression is regulated by several posttranslationalmechanisms. LIGHT is cleaved from the cell surface by matrixmetalloproteinases within minutes of its expression and accumulates as asoluble molecule (isoform 1; represents approximately residues 83-240;Swiss-Prot O43557-1). The cell surface cytoplasmic segment representsisoform 2 (Swiss-Prot O43557-2). Additionally, various cell types storeLIGHT in vesicles and excrete them upon activation by variousphysiological stimuli. Although the role of the soluble form of LIGHT isnot well characterized, it may serve as a negative feedback loop toinhibit the function of the membrane-bound form by competing for HVEMand LTβR.

LIGHT interacts with three different receptors: (1) herpesvirus entrymediator (HVEM) on T cells, (2) LTβR which is expressed primarily onepithelial and stromal cells, and (3) the soluble decoy receptor 3 onvarious cells. These interactions endow LIGHT with different functions.Interaction with LTβR on stromal cells is associated with the productionof various cytokines/chemokines, lymph node (LN) organogenesis, andrestoration of secondary lymphoid structures. On the other hand,interaction of LIGHT with HVEM receptor on lymphocytes results inactivation and production of cytokines, dominated by IFN-γ and GM-CSF.In this context, the LIGHT/HVEM axis appears to deliver costimulatorysignals associated with the activation of Th1 type responses which playcritical roles in tumor eradication.

LIGHT plays a role in lymphoid organogenesis and in the generation ofTh1 type responses. See, e.g., Yang et al., 2002, J. Biol. Regul.Homeost. Agents, 16:206-10; Schneider et al., 2004, Immunol. Rev.,202:49-66.

The effect of LIGHT has been shown in different tumor models both invitro and in vivo. Chronic lymphocytic leukemic cells transduced byherpes simplex virus amplicon expressing LIGHT have been reported toenhance T cell proliferation in mixed lymphocyte reactions.Over-expression of LIGHT on MDA-MB-231 human breast cancer cells hasbeen shown to suppress tumor growth. Transfection of LIGHT intodifferent cancer cell lines stimulate ICAM-1 expression in these cells.The presence of ICAM-1 is believed to be beneficial as it enableseffective signaling to produce antitumor activity in tumor cells.Another important function of LIGHT, besides T cell activation, is itsability to transduce signals through LTβR, which plays an important rolein the development of secondary lymphoid structures mediated through theinduction of chemokine expression as well as adhesion molecules instromal cells. The interaction of LIGHT with LTβR on stromal cellsregulates the expression of CCL21, which control the homing of naïve Tcells to lymphoid tissues.

One advantage of embodiments of the invention where the immuneco-stimulatory polypeptide is LIGHT is the ability of LIGHT to stimulatelymphoid organogenesis and support the generation of Th1 type responses.Another advantage is the ability of LIGHT to stimulate immune responsesagainst tumors and activate the tumor stroma to further augment theseresponses.

The stroma serves as a physical barrier to prevent lymphocyteinfiltration into the tumor site. The stroma also inhibits lymphocyteactivation within the tumor microenvironment. This may be due to thelack of costimulatory signals needed for T cell activation and/or thepresence of various immunoinhibitory soluble mediators, such as TGF-βand IL-10, that are synthesized and secreted by both stromal fibroblastsand tumor cells. The stroma promotes immunological ignorance byconfining tumor cells to the tumor site, thereby preventing them fromtrafficking to the regional lymph nodes.

Tumor stromal cells also express various immunological receptors, suchas LTβR, that can be exploited for the enhancement of anti-tumorimmunity in accordance with the present invention.

4. OX40L

OX40L is expressed by dendritic cells and other APC and binds to OX40which is present on activated T cells. OX40L contains 183 amino acids(21950 Da). See Miura et al. Mol. Cell. Biol. 11:1313-1325 (1991). Thefull amino acid sequence of OX40L can be found under accession no.P23510 in the Swiss-Prot database. OX40L is a type II glycoprotein witha cytoplasmic domain at residues 1-23, a transmembrane domain atresidues 24-50 and an extracellular domain at residues 51-183. Thenucleotide sequence of OX40L is 3510 bp, with the coding sequence being157-708 (see Genbank accession no. NM_(—)003326.2). Residues 51-183, orfragments thereof of OX40L that can bind to its cognate receptor OX40,can be linked or expressed as a C-terminal fusion to a binding pairmember for use in accordance with the present invention.

5. CD40L

CD40L is expressed by activated T cells and also exists as anextracellular soluble form which derives from the membrane form byproteolytic processing. CD40L (a.k.a. TNFSF5) contains 261 amino acids(29350 Da). See Villinger et al. Immunogenetics 53:315-328 (2001). Thefull amino acid sequence of CD40L can be found under accession no.Q9BDN3. CD40L is a type II glycoprotein with a cytoplasmic domain atresidues 1-22, a transmembrane domain at residues 23-43 and anextracellular domain at residues 44-261. The nucleotide sequence ofCD40L is 1834 bp, with the coding sequence being 73-858 (see Genbankaccession no. NM_(—)000074). Residues 44-261, or fragments thereof ofCD40L that can bind to its cognate receptor CD40, can be linked orexpressed as an N-terminal fusion to a binding pair member for use inaccordance with the present invention.

6. PD-L1

PD-L1 is expressed on activated T and B cells, dendritic cells,keratinocytes and monocytes. PD-L1 (a.k.a. B7-H; B7H1; PDL1; PDCD1L1)contains 290 amino acids (33275 Da). See Dong et al. Nat. Med. 5:1365-1369 (1999). The full amino acid sequence of PD-L1 can be foundunder accession no. Q9NZQ7 in the Swiss-Prot database. PD-L1 contains290 amino acids of which 18 amino acids at the N terminus represent thesignal sequence. The extracellular domain is located at amino acids19-238, a transmembrane domain is located at resides 239-259 and acytoplasmic domain is located at residues 260-290. The nucleotidesequence of PD-L1 (1553 bp) is available in public databases (seeGenbank accession no. NM_(—)014143) (coding sequence is 53-925).Isoforms of PD-L1 exist by way of alternative splicing. Theextracellular domain or fragments thereof of PD-L1 that can bind to itscognate receptor PDCD1, can be linked or expressed as an N-terminalfusion to a binding pair member for use in accordance with the presentinvention.

7. GL50

GL50 isoform 1 is widely expressed (brain, heart, kidney, liver, lung,pancreas, placenta, skeletal muscle, bone marrow, colon, ovary,prostate, testis, lymph nodes, leukocytes, spleen, thymus and tonsil);GL50 isoform 2 (swissprot O75144) is expressed in lymph nodes,leukocytes and spleen and on activated monocytes and dendritic cells.GL50 (a.k.a. B7-H2; B7H2; B7RP-1; B7RP1; ICOS-L; ICOSLG; KIAA0653; andLICOS) contains 290 amino acids (33275 Da). See Wang et al. Blood96:2808-2813 (2000). The full amino acid sequence of GL50 can be foundunder accession no. O75144 in the Swiss-Prot database. GL50 contains 302amino acids of which 18 amino acids at the N terminus represent thesignal sequence. The extracellular domain is located at amino acids19-256, a transmembrane domain is located at resides 257-277 and acytoplasmic domain is located at residues 278-302. The nucleotidesequence of GL50 (3239 bp) is available in public databases (see Genbankaccession no. NM_(—)015259) (coding region representing 135-1043).Isoforms of GL50 exist by way of alternative splicing. The extracellulardomain or fragments thereof of GL50 that can bind to its cognatereceptor ICOS, can be linked or expressed as an N-terminal fusion to abinding pair member for use in accordance with the present invention.

Table 1 summarizes various exemplary costimulatory molecules and theirreceptors and includes embodiments of coreceptor ligand pair conjugates.

TABLE 1 Construct name and orientation Receptor Receptor expressionCD80-CSA CD28 Constitutive on almost all human CD4 T cells andapproximately 50% of CD8 T cells GL50-CSA ICOS Detectable on resting Tcells Upregulated on activated CD4⁺ T and CD8⁺ T cells and NK cellsPD-L1-CSA PD-1 Inducible on CD4⁺ and CD8⁺ T cells, B cells, andmonocytes Low levels on NK-T cells CSA-CD40L CD40 Constitutive on Bcells, monocytes, DC, endothelial and epithelial cells CSA-4-1BBL CD137Inducible on activated T cells (peak 48 h, decline 96 h) as well ascytokine-treated NK cells Constitutive on subsets of DC (low), humanmonocytes, follicular DC, CD4⁺ CD25⁺ regulatory T cells. CSA-OX40L OX40Inducible on activated CD4 (preferentially) and CD8 (strong antigenresponse) T cells (peak 48 h, decline 96 h) CSA-LIGHT HVEM Constitutiveon resting T cells, monocytes, and immature DC Downregulated upon T cellactivation and DC maturation

Other immune co-stimulatory polypeptides can be used in accordance withthe invention. For example US 2003/0219419 (the entire contents of whichare incorporated herein by reference in their entirety) describesIL-2-CSA fusion proteins, and CSA-CD40L fusion proteins that are usefulin the present invention. In summary, exemplary immune co-stimulatorypolypeptides useful in accordance with the present invention include thefollowing.

TABLE 2 B7 and CD28 FAMILY MEMBERS LIGAND RECEPTOR CD80 (B7.1) CD28,CTLA-4 (CD152) CD86 (B7.2) CD28, CTLA-4 ICOSL (B7h, B7-H2, B7RP-1, GL50,LICOS) ICOS (AILIM) PD-L1 (B7-H1) PD-1 PD-L2 (B7-DC) PD-1 B7-H3 UnknownB7-H4 (B7x; B7S1) Unknown (BTLA?) Unknown (HVEM*) BTLA *it is a TNFmember

TABLE 3 TNF FAMILY MEMBERS LIGAND RECEPTOR OX40L OX40 (CD134) 4-1BBL4-1BB (CD137) CD40L (CD154) CD40 CD27L (CD70) CD27 CD30L CD30 LIGHTHVEM, LTβR, DcR3 GITRL GITR BAFF (BLyS)** BAFF-R, TACI, BCMA APRIL**TACI, BCMA **these are B cell related

TABLE 4A References for nucleotide and/or amino acid sequences of B7Family Members LIGAND (Human) REFERENCE CD80 (B7.1) Freeman et al., J.Immunol. 143: 2714-2722(1989). CD86 (B7.2) Freeman et al., Science 262:909-911(1993). ICOSL Wang et al., Blood 96: 2808-2813(2000). Yoshinagaet al., Int. Immunol. 12: 1439-1447(2000). PD-L1 Dong et al., Nat. Med.5: 1365-1369(1999). Freeman et al., J. Exp. Med. 192: 1027-1034(2000).PD-L2 Tseng et al., J. Exp. Med. 193: 839-846(2001). Latchman et al.,Nat. Immunol. 2: 261-268(2001). B7-H3 Steinberger et al., Submitted(SEP-2003) to EMBL/GenBank/DDBJ databases. Mingyi et al., J. Immunol168: 6294-6297(2002). B7-H4 Zang et al., Proc. Natl. Acad. Sci. U.S.A.100: 10388-92(2003). (B7x; B7S1) Sica et al., Submitted (APR-2003) toEMBL/GenBank/DDBJ databases.

TABLE 4B References for nucleotide and/or amino acid sequences of TNFFamily Members LIGAND REFERENCE OX40L Baum et al., Circ. Shock 44:30-34(1994). Miura et al., Mol. Cell. Biol. 11: 1313-1325(1991). Godfreyet al., J. Exp. Med. 180: 757-762(1994). 4-1BBL Alderson et al., Eur. J.Immunol. 24: 2219-2227(1994). CD40L Graf et al., Eur. J. Immunol. 22:3191-3194(1992). Hollenbaugh et al., EMBO J. 11: 4313-4321(1992). CD27LGoodwin et al., Cell 73: 447-456(1993). (CD70) CD30L Smith et al., Cell73: 1349-1360(1993). LIGHT Mauri et al., Immunity 8: 21-30(1998). GITRLGurney et al., Curr. Biol. 9: 215-218(1999). BLyS Moore et al., Science285: 260-263(1999). APRIL Hahne et al., J. Exp. Med. 188:1185-1190(1998).

Antigens & Infectious Agents

The methods and compositions of the invention are useful for generatingor enhancing an immune response against any antigen or infectious agent,including TAAs, antigens associated with an infectious agent, and aninfectious agent itself. In accordance with the invention, an antigenassociated with the targeted tumor or infectious agent (or theinfectious agent itself) is presented to immune cells, therebygenerating or enhancing an immune response.

1. TAAs

In one embodiment, the antigen is a TAA, and the invention providescancer immunotherapy methods effective to generate or enhance apatient's immune response against a tumor. In accordance with thisembodiment, the invention provides methods of reducing tumor size andmethods of inhibiting the growth of tumor cells.

Representative tumor cells against which this invention is usefulinclude, without limitation, carcinomas, which may be derived from anyof various body organs including lung, liver, breast, bladder, stomach,colon, pancreas, skin, and the like. Carcinomas may includeadenocarcinoma, which develop in an organ or gland, and squamous cellcarcinoma, which originate in the squamous epithelium. Other cancersthat can be treated include sarcomas, such as osteosarcoma or osteogenicsarcoma (bone), chondrosarcoma (cartilage), leiomyosarcoma (smoothmuscle), rhabdomyosarcoma (skeletal muscle), mesothelial sarcoma ormesothelioma (membranous lining of body cavities), fibrosarcoma (fibroustissue), angiosarcoma or hemangioendothelioma (blood vessels),liposarcoma (adipose tissue), glioma or astrocytoma (neurogenicconnective tissue found in the brain), myxosarcoma (primitive embryonicconnective tissue), an esenchymous or mixed mesodermal tumor (mixedconnective tissue types). In addition myelomas, leukemias, and lymphomasare also susceptible to treatment.

A number of TAAs associated with specific tumor types have beenidentified. These include human telomerase reverse transcriptase(hTERT), survivin, MAGE-1, MAGE-3, human chorionic gonadotropin,carcinoembryonic antigen, alpha fetoprotein, pancreatic oncofetalantigen, MUC-1, CA 125, CA 15-3, CA 19-9, CA 549, CA 195,prostate-specific antigens; prostate-specific membrane antigen,Her2/neu, gp-100, mutant K-ras proteins, mutant p53, truncated epidermalgrowth factor receptor, chimeric protein ^(p210)BCR-ABL; E7 protein ofhuman papilloma virus, and EBNA3 protein of Epstein-Barr virus. Any ofthese antigens, antigenic fragments thereof, and mixtures of antigensand/or fragments can be used in accordance with the invention togenerate or enhance a patient's anti-tumor immune response. Table 5lists some exemplary TAAs and diseases associated with such TAAs.

TABLE 5 Antigen Diseases cTAGE-1 and variants Cutaneous T cell lymphomaBLA or globotriaosylceramide Burkitt's lymhoma (P^(k) antigen) humanT-cell leukemia virus-associated Adult T-cell cell membrane antigens(HTLV-MA) leukemia'lymphoma (ATL) Thymocyte surface antigen JL1 Majorityof acute leukemias Adult T cell leukemia associated, human Adult T cellleukemia retrovirus associated antigen (ATLA) Epstein-Barr virus (EPV)antigens Burkitt's lymphoma, Hodgkin's disease Anaplastic lymphomakinase (ALK), CD30+ anaplastic large fusion proteins (NPM/ALK andvariants) cell lymphoma (ALCL) Common acute lymphoblastic leukemia Mostacute lymphoblastic antigen (CALLA) leukemias Immunoglobulin Id; Type IILymphoproliferative diseases glycoproteins (e.g., HM1.24; KW-2, KW-4,KW-5, KW-12); Oncofetal antigen immature laminin receptor protein(OFA-iLRP); EBV proteins (e.g., LMP2A)

Additional human TAAs recognized by T-cells may be found in, forexample, Novellino et al. “A listing of human tumor antigens recognizedby T cells: March 2004 update” Cancer Immunology and Immunotherapy, 54:187-207 (2005) which is incorporated by reference herein. Many animalTAAs corresponding to animal correllaries of these diseases, and toother animal diseases, are known in the art and also included within thescope of the invention.

In one embodiment of the invention, the TAA is selected from the groupconsisting of human telomerase reverse transcriptase (hTERT) andsurvivin as TAAs. hTERT is expressed in >85% of human cancers, while itsexpression is restricted in normal tissues. See, e.g., Vonderheide etal., Immunity 1999, 10: 673-79. Similarly, survivin, which has beenidentified as an inhibitor of apoptosis, is absent from normal tissuesbut expressed in most tumor types including lung, colon, pancreas,prostate and breast cancer. See, e.g., Ambrosini et al., Nat. Med. 1997,3: 917-21. Because these TAAs are expressed in the majority of cancertypes and are rare or absent from normal tissues, they are attractiveantigens for use in cancer immunotherapy methods according to thepresent invention.

In another embodiment of the invention, the TAA is associated withcervical cancer. Approximately 500,000 women worldwide develop cervicalcancer yearly and it is the second leading cause of death from cancer inwomen. Cervical cancer has been directly linked to genital viralinfection by human papillomavirus (HPV) and is a worldwide healthproblem. HPV type 16 in particular is found in roughly half of cervicalcancers. Genital HPV types 16 and 18, and less frequently, types 31, 33,35, 45, 51 and 56, also have been implicated in the etiology of cervicaland other anogenital cancers. The HPV types found in cancer cells havetransforming activity in in vitro studies and the viral transformingproteins, E6 and E7 (also known as “early” proteins), are consistentlyexpressed in cervical cancer cell lines and in HPV-associated cancers.E6 and E7 are known to bind the tumor suppressors, p53 andretinoblastoma (Rb), respectively. In HPV-associated malignanttransformation, late genes (L1 and L2) and some early genes (E1 and E2)are usually lost, leaving E6 and E7 as the only open reading framesfrequently found in carcinomas. Expression of E6 and E7 is likely toovercome the regulation of cell proliferation normally mediated byproteins like p53 and Rb, allowing uncontrolled growth and providing thepotential for malignant transformation.

Thus, in accordance with one specific embodiment of the invention, theTAA is one or more of E6 and E7. The use of E6 and E7 in accordance withthe invention may offer several advantages. First, E6 and E7 areconsistently expressed in most cervical cancers. Second, while mosttumor antigens are derived from normal proteins or mutated self-protein,E6 and E7 are completely foreign viral proteins, and may harbor moreantigenic peptides or epitopes than a mutant protein. Third, E6 and E7play an important role in the induction and maintenance of the malignantphenotype, and without functional E6 and E7, these cells would cease tobe tumorigenic.

The nucleotide and amino acid sequences of the E6 and E7 proteins fromdifferent species (e.g., human, bovine) and for different papillomavirustypes (e.g., HPV16 and 18) are known in the art. See, e.g., the HPVsequence database athttp://www.stdgen.lanl.gov/stdgen/virus/hpv/index.html. The amino acidsequences of HPV16 E6, an HPV16 E6 variant, and E7 are set forth inFIGS. 6A (SEQ ID NO:11), 6B (SEQ ID NO:12) and 6C (SEQ ID NO:13),respectively.

2. Infectious Agents

Representative infectious agents against which this invention is usefulinclude, without limitation, any virus, bacteria, fungi or protozoan.Table 6 lists examples of infectious agents.

TABLE 6 ETIOLOGICAL ASSOCIATED AGENT GENUS DISEASE BACTERIALMycobacterium Tuberculosis tuberculosis Bacillus anthracis AnthraxStaphylococcus Sepsis aureus VIRAL Adenoviridae Mastadenovirus Ifectious canine hepatitis Arenaviridae Arenavirus Lymphocyticchoriomeningitis Caliciviridae Norovirus Norwalk virus infectionCoronaviridae Coronavirus Severe Acute Respiratory Syndrome TorovirusFiloviridae Marburgvirus Viral hemorrhagic fevers Ebolavirus Viralhemorrhagic fevers Flaviviridae Flavivirus West Nile EncephalitisHepacivirus Hepatitis C virus infection Pestivirus Bovine VirusDiarrhea, Classical swine fever Hepadnaviridae OrthohepadnavirusHepatitis Herpesviridae Simplexvirus cold sores, genital herpes, bovinemammillitis Varicellovirus chickenpox, shingles, abortion in horses,encephalitis in cattle Cytomegalovirus infectious mononucleosisMardivirus Marek's disease Orthomyxoviridae Influenzavirus A InfluenzaInfluenzavirus B Influenza Papillomaviridae Papillomavirus Skin warts,skin cancer, cervical cancer Picornaviridae Enterovirus Polio RhinovirusCommon cold Aphthovirus Foot-and-mouth disease Hepatovirus HepatitisPoxviridae Orthopoxvirus Cowpox, vaccinia, smallpox ReoviridaeRotaviruses Diarrhea Orbivirus Blue tongue disease RetroviridaeGammaretrovirus Feline leukemia Deltaretrovirus Bovine leukemiaLentivirus Human immunodeficiency, FIV, and SIV Rhabdoviridae LyssavirusRabies Ephemerovirus Bovine ephemeral fever Togaviridae AlphavirusEastern and Western equine encephalitis PARASITIC Plasmodium MalariaLeishmania Leishmaniasis FUNGAL Aspergillis Candida Coccidia CryptococciGeotricha Histoplasma Microsporidia Pneumocystis

Human and avian influenza, HIV, hepatitis C, tuberculosis, west nilevirus, cryptococosis (meningitis) herpes, chlamydia, and anthrax arerepresentative of infectious agents. Any antigen associated with theinfectious agent can be used in accordance with the invention.

In accordance with one embodiment, the infectious agent itself is usedin a conjugate according to the invention. In accordance with thisembodiment, a conjugate comprising the infectious agent, such as avirus, and a binding pair member is used. Any infectious agent may beused, such as a virus, including a human or avian influenza virus orHIV, or any other virus. The infectious agent may be modified orattenuated to reduce or eliminate its infectivity.

For the purpose of illustration only, this aspect of the invention isdescribed in more detail with reference to influenza. Influenza is acontagious disease caused by the influenza virus, and affects therespiratory tract, often resulting in symptoms in the nose, throat andlungs, as well as fever, headache, tiredness and aches. It can also leadto complications such as pneumonia, bronchitis, or sinus and earinfections or exacerbate chronic conditions. Influenza viruses areclassified as type A, B or C. Strains belonging to types A and Bcirculate in the population and are associated with most cases of humaninfluenza. Type A influenza causes the overwhelming majority of publichealth problems in humans.

Type A influenza viruses are subtyped depending on the composition oftwo of its proteins; hemagglutinin (H), a protein that facilitatesbinding and entry of the virus into the target cell, and neuraminidase(N), a protein involved in the release of newly formed virus particlesfrom infected cells and spreading it through the body. Fifteenhemagglutinin subtypes (H1-H15) and 9 neuraminidase subtypes (N-1-N9)have been identified. Large outbreaks of influenza in humans have beencaused by three hemagglutinin subtypes (H1, H2 and H3) and twoneuraminidase subtypes (N1 and N2). For example, the hemagglutinin ofthe 1918 flu virus was H1, its neuraminidase was N1, so it is designatedas an H1N1 subtype. Other outbreaks have included the H2N2 subtype in1957, H3N2 in 1968, and H5N1 in the recent outbreaks in birds and humansin Southeast Asia, China, and now Europe and the Middle East.

Influenza A viruses constantly evolve by mechanisms which involvemutations or changes in the reactive or antigenic sites of hemagglutininand neuraminidase, or by the sudden replacement of one hemagglutinin orneuraminidase subtype by another subtype. These mechanisms result in newvirus subtypes and allow the influenza virus to evade the defenses ofthe immune system and spread. Antigenic variants of influenza A virusesemerge every year and demand an updated vaccine formulation based onongoing international surveillance of influenza virus by the WorldHealth Organization. Due to this phenomenon in which new influenza virussubtypes constantly emerge, such as H5N1 in recent years, more majoroutbreaks of influenza are expected to occur. In certain plausiblebioterrorism scenarios, laboratory-derived viruses would similarly bedesigned to effect antigenic changes and hence to cause outbreaks thatwould evade established host defenses.

The conjugates of the present invention can be used in influenzavaccines that are easy to produce and manufacture quickly, whoseantigenic component can be changed and updated based on the currenthealth needs without difficulty, that selectively targets viralmachinery and infected cells, and that can be administeredpost-infection for a therapeutic effect as well as pre-infection forprevention.

Thus, in accordance with one embodiment, an influenza antigen (orantigenic fragment thereof) is used as the antigen component of aconjugate of the present invention. For example, the antigen maycomprise one or more of H1 and N1 (both highly immunogenic) and/or oneor more of nucleoprotein (NP) and matrix protein 1 (MP1) and/or matrixprotein 2 (MP2) (all highly conserved, structural proteins). Proteinsfrom pandemic strains such as H5, also can be used as antigens inaccordance with the invention. While not wanting to be bound by anytheory, intracellular proteins such as NP and MP2 may provide a moreuniversal vaccine because they exhibit little or no variance andtherefore may prevent heterologous viral infections without the need forannual adjustment. For example, NP exhibits>90% protein sequencehomology among influenza A isolates and contains dominant cytotoxic Tcell target epitopes. Other influenza antigens useful in the presentinvention include PA, PB1 and PB2 (RNA polymerase subunits) and NS1 andNS2 (interferon response inhibitor and RNP nuclear export). See also,Brown, 2000, Biomed. Pharmacother. 54: 196-209; Steinhauer et al., 2002,36: 305-32; De Jong et al., 2000, 40: 218-28; Alexander, Vet. Microbiol.74: 3-13.

Thus, in accordance with one embodiment, a Type A influenzahemagglutinin protein (or antigenic fragment thereof) is used as theantigen component of a conjugate of the present invention. Currently theprevention of influenza is achieved by subcutaneous injection of aninfluenza vaccine with H as the major component. For example, H1 frominfluenza virus A/PuertoRico/8/34 (PR8) (H1N1) is the predominant,circulating H protein and has been well-characterized, and can be usedin accordance with the invention. In another embodiment, a Type Ainfluenza neuraminidase protein (or antigenic fragment thereof) is usedas the antigen component of the conjugate. A composition comprisingeither an H protein-containing conjugate or an N protein-containingconjugate is useful as a vaccine against influenza. (For example,current influenza vaccines comprise an H protein as the major component,and have been shown to induce sufficient immunity to prevent an epidemicof homologous virus.) Alternatively, it may be advantageous toadminister both a conjugate comprising an H protein and a conjugatecomprising an N protein, or any combination of antigens, such as acombination of variable and conserved antigens. This could be effectedby administering two or more compositions, each comprising a singleantigen-containing conjugate, or by providing two or more conjugates(and, therefore, two or more antigens) in a single composition. In oneembodiment, the antigen components of the conjugate(s) are chosen basedon current public health needs.

In one embodiment, the conjugate comprises 4-1BBL as the immuneco-simulatory polypeptide. While not wanting to be bound by any theory,it is believed that this conjugate, when administered to a patient invivo, will bind to DCs through the interaction between 4-1BBL and the4-1BB receptor on DCs, resulting in internalization of the vaccine andpresentation of the influenza antigen on the surface of the DC, as wellas activation and maturation of DCs. Activation of the DC may in turnlead to interaction with and activation of CD8 and CD4 T cells.Activated CD4 T cells may then interact with B cells and cause theiractivation and differentiation into antibody secreting cells, leading toa humoral response. Activation of CD8 T cells also may lead todifferentiation and proliferation of more CD8 T cells which function tokill virus-infected cells. The conjugate also may bind directly toactivated CD8 and CD4 T cells expressing the 4-1BB receptor, furtheramplifying the signal. In addition, the conjugate (via 4-1BBL) may binddirectly to activated natural killer (NK) cells, which also function tokill virus-infected cells, all of which results in a more robust immuneresponse. Thus, the conjugate will generate both cellular and humoralresponses that support its efficacy in therapeutic and prophylacticvaccines.

The antigen components of conjugates useful as vaccines against otherinfectious agents can be selected in an analogous manner by thoseskilled in the art, based on the antigens associated with thoseinfectious agents. For example, antigens associated with HIV include HIVenvelope gp120 epitopes (e.g, variable loops such as V3), or other HIVproteins such as Gag proteins (Pr55^(gag), matrix p17, capsid p24,nucleocapsid p7), p5; Pol (polymerase), Vif (viral infectivity factorp23); Vpr (viral protein R p15); Rev (regulator of viral gene expressionp19); Vpu (viral protein U); Env (gp 160, gp120, gp41); Tat(trancripinal activator p14); and Nef (gegative effector p24). See,e.g., Peters, 201, Vaccine 2: 688-705; Michael, 2003, Clin. Med. 3:269-72; Gandhi & Walker, 2002, Ann. Rev. Med. 53: 149-72; Haseltine,1991, FASEB 5: 2349-60. Other antigens useful in vaccines includecapsular polysaccharides of Haemophilus influenzae type b, capsusalrpolysaccharides of Neisseria meningitidis, capsusalr polysaccharides ofStreptococcus pneumoniae, surface antigens of Hepatitis B, andinactivated exotoxins of diphtheria and tetanus toxins. These antigenscan be used in accordance with the present invention as described abovewith reference to influenza antigens.

Binding Pairs

An exemplary binding pair is biotin and streptavidin (SA) or avidin. SAor avidin fragments which retain substantial binding activity forbiotin, such as at least 50% or more of the binding affinity of nativeSA or avidin, respectively, also may be used. Such fragments include“core streptavidin” (“CSA”) a truncated version of the full-lengthstreptavidin polypeptide which may include streptavidin residues 13-138,14-138, 13-139 and 14-139. See, e.g., Pahler et al., J. Biol. Chem.1987, 262:13933-37. Other truncated forms of streptavidin and avidinthat retain strong binding to biotin also may be used. See, e.g. Sano etal., J Biol. Chem. 1995 November 24, 270(47): 28204-09 (describing corestreptavidin variants 16-133 and 14-138) (U.S. Pat. No. 6,022,951).Mutants of streptavidin and core forms of strepavidin which retainsubstantial biotin binding activity or increased biotin binding activityalso may be used. See Chilcoti et al., Proc Natl Acad Sci USA. 1995 Feb.28; 92(5):1754-8; Reznik et al., Nat. Biotechnol. 1996 August;14(8):1007-11. For example, mutants with reduced immunogenicity, such asmutants mutated by site-directed mutagenesis to remove potential T cellepitopes or lymphocyte epitopes, can be used. See Meyer et al., ProteinSci. 2001 10: 491-503. Likewise, mutants of avidin and core forms ofavidin which retain substantial biotin binding activity or increasedbiotin binding activity also may be used. See Hiller et al., J. Biochem.(1991) 278: 573-85; Livnah et al. Proc Natl Acad Sci USA (0: 5076-80(1993). For convenience, in the instant description, the terms “avidin”and “streptavidin” as used herein are intended to encompassbiotin-binding fragments, mutants and core forms of these binding pairmembers. Avidin and streptavidin are available from commercialsuppliers. Moreover, the nucleic acid sequences encoding streptavidinand avidin and the streptavidin and avidin amino acid sequences can befound, for example, in GenBank Accession Nos. X65082; X03591;NM_(—)205320; X05343; Z21611; and Z21554.

As used herein “biotin” includes biotin-containing moieties that areable to bind to surfaces, such as cell surfaces (including tumor cellsurfaces), such as NHS-biotin and EZ-Link™ Sulfo-NHS-LC-Biotin (Pierce).Such protein reactive forms of biotin are available commercially.

The interaction between biotin and its binding partner, avidin orstreptavidin, offers several advantages in the context of the presentinvention. For example, biotin has an extremely high affinity for bothstreptavidin (10¹³ M⁻¹) and avidin (10¹⁵ M⁻¹). Additionally, bothstreptavidin and avidin are tetrameric polypeptides that each bind fourmolecules of biotin. Immune co-stimulatory moieties comprisingstreptavidin or avidin therefore have a tendency to form tetramers andhigher structures. As a result, they can cross-link their correspondingimmune cell receptors for more potent signal transduction, such asthrough aggregation of receptors.

Those skilled in the art will recognize that other mechanisms (e.g.,other conjugation methods using, for example, other linking moieties orchemical or genetic cross-linking) can be used to provide higher-orderstructures of immune co-stimulatory molecules, such as conjugatescomprising dimers, trimers, tetramers and higher-order multimers ofimmune co-stimulatory molecules, which also will exhibit advantageousproperties. Such conjugates are included within the scope of thisinvention.

Conjugates

A conjugate comprising an immune co-stimulatory polypeptide, antigen, orinfectious agent and a member of a binding pair can be made by methodswell known in the art. For example, the polypeptide/antigen/infectiousagent and binding pair member can be covalently bound to each other orconjugated to each other directly or through a linker. In accordancewith one embodiment, the polypeptide/antigen/infectious agent andbinding pair member are components of a fusion protein. Fusion proteinscan be made by any of a number of different methods known in the art.For example, one or more of the component polypeptides of the fusionproteins can be chemically synthesized or can be generated using wellknown recombinant nucleic acid technology. (As used herein, “nucleicacid” refers to RNA or DNA.) Nucleic acid sequences useful in thepresent invention can be obtained using, for example, the polymerasechain reaction (PCR). Various PCR methods are described, for example, inPCR Primer: A Laboratory Manual, Dieffenbach 7 Dveksler, Eds., ColdSpring Harbor Laboratory Press, 1995.

In accordance with one embodiment, an immune co-stimulatory polypeptideis bound via its C-terminus to the N-terminus the binding pair member.For example, an immune co-stimulatory polypeptide can be bound via itsC-terminus to the N-terminus of core streptavidin (CSA). Thus theinvention includes CD80-CSA fusion proteins, where the CD80 moiety isbound via its C-terminal to the N-terminal of CSA. In accordance withanother embodiment, the immune co-stimulatory polypeptide is bound viaits N-terminus to the C-terminus of the binding pair member. Forexample, an immune co-stimulatory polypeptide can be bound via itsN-terminus to the C-terminus of CSA. For example, the invention includesCSA-4-1BBL, CSA-LIGHT, CSA-CD40L, and CSA-OX40L fusion proteins, wherethe CSA moiety is bound via its C-terminal to the N-terminal of theimmune co-stimulatory polypeptide. The immune co-stimulatory polypeptidemay be directly bound to the binding pair member or may be bound via oneor more linking moieties, such as one or more linking polypeptides.

In accordance with one embodiment, the immune co-stimulatorypolypeptide, antigen or infectious agent is biotinylated. Biotinylatedconjugates can be made by methods known in the art, and exemplifiedbelow in the examples.

For example, Biotin AviTag technology from Avidity, Inc. (Denver, Colo.)can be used to generate biotinylated proteins or infectious agents. TheBiotin AviTag is comprised of a unique 15 amino acid peptide that isrecognized by biotin ligase, BirA, that attaches biotin to a lysineresidue in the peptide sequence. Schatz, 1993, Biotechnology, 11:1138-43. The Biotin AviTag can be genetically fused to any protein ofinterest, allowing the protein to be tagged with a biotin molecule.

One potential drawback to the Biotin AviTag technology is thepossibility of a low degree of biotinylation, because the systembiotinylates the protein at a single, unique lysine residue in the tagregion. To overcome any such problem, the purified tagged proteins canbe modified in vitro using purified biotin ligase. Because thebiotinylation is performed enzymatically, the reaction conditions aregentler, the labeling is highly specific, and the reaction is moreefficient than chemical modification of the protein using biotinderivatives. Alternatively, the methods described in Jordan, et al,2003, Clin. Diag. Lab. Immunol. 10: 339-44, can be used to produce agenetically engineered biotinylated protein.

Fragments of an immune co-stimulatory polypeptide, binding pair memberantigen, or infectious agent are useful in the present invention, aslong as the fragment retains the activity of the referent full-lengthmoiety. Thus, for example, an immune co-stimulatory fragment shouldretain its immune co-stimulatory activity (e.g., retain its ability tobind its receptor or ligand), a binding member fragment should retainits ability to bind with its binding partner, and an antigen orinfectious agent fragment should retain its ability to induce an immuneresponse against the referent full-length antigen or infectious agent.Fragments can be screened for retained activity by methods that areroutine in the art. Exemplary fragments of immune co-stimulatorypolypeptides are set forth above.

The conjugates may include a linker such as a peptide linker between thebinding pair member and the immune co-stimulatory polypeptide, antigen,or infectious agent. The linker length and composition may be chosen toenhance the activity of either or both functional ends of the conjugate(e.g., co-stimulatory polypeptide/antigen.infectious agent or bindingpair member). The linker is generally from about 3 to about 15 aminoacids long, more preferably about 5 to about 10 amino acids long,however, longer or shorter linkers may be used or the linker may bedispensed with entirely. Flexible linkers (e.g. (Gly₄Ser)₃) such as havebeen used to connect heavy and light chains of a single chain antibodymay be used in this regard. See Huston, et al. (1988) Proc. Nat. Acad.Sci. USA, 85:5879-5883; U.S. Pat. Nos. 5,091,513, 5,132,405, 4,956,778;5,258,498, and 5,482,858. Other linkers are FENDAQAPKS or LQNDAQAPKS.One or more domains of and immunoglobulin Fc region (e.g CH1, CH2 and/orCH3) also may be used as a linker. Chemical linkers also may be used.

Nucleic acids and polypeptides that are modified, varied, or mutatedalso are useful in the present invention, as long as they retain theactivity of the referent nucleic acid or polypeptide. For example,nucleic acid and polypeptide sequences suitable for use in the presentinvention can have at least about 80% sequence identity (including atleast 80% sequence identity) to a referent nucleic acid or polypeptide,i.e., to a nucleic acid encoding a known immune co-stimulatorypolypeptide or binding pair member. In some embodiments, the nucleicacid sequence or polypeptide has at least about 85%, at least about 90%,at least about 95%, or at least about 99% sequence identity to thereferent nucleic acid or polypeptide.

The invention encompasses nucleic acids with base changes that are“silent,” in that they encode the same amino acid (i.e. degeneratenucleic acid sequences). The invention also encompasses nucleic acidsthat encode polypeptides with conservative amino acid substitutions, andsuch polypeptides. Conservative amino acid substitutions (for example,substituting one hydrophobic residue with a different hydrophobicresidue) are well known in the art and can be effected, e.g., by pointmutations and the like. The suitability of a given modified sequence,variant or mutant can be confirmed using receptor binding and/orbiological screening methods that are known in the art, such as thosediscussed above with reference to fragments.

As used herein, “% sequence identity” is calculated by determining thenumber of matched positions in aligned nucleic acid or polypeptidesequences, dividing the number of matched positions by the total numberof aligned nucleotides or amino acids, respectively, and multiplying by100. A matched position refers to a position in which identicalnucleotides or amino acids occur at the same position in the alignedsequences. The total number of aligned nucleotides or amino acids refersto the minimum number of nucleotides or amino acids that are necessaryto align the second sequence, and does not include alignment (e.g.,forced alignment) with non-homologous sequences, such as those that maybe fused at the N-terminal or C-terminal of the sequence of interest(i.e., the sequence encoding the immune co-stimulatory polypeptide orbinding pair member). The total number of aligned nucleotides or aminoacids may correspond to the entire coding sequence or may correspond tofragments of the full-length sequence as defined herein.

Sequences can be aligned using the using the algorithm described byAltschul et al. (1997, Nucleic Acids Res., 25:3389-3402) as incorporatedinto the BLAST (basic local alignment search tool) programs, availableat ncbi.nlm.nih.gov on the World Wide Web. BLAST searches or alignmentscan be performed to determine percent sequence identity between anucleic acid molecule (the “query sequence”) and any other sequence orportion thereof using the Altschul algorithm. BLASTN can be used toalign and compare the identity between nucleic acid sequences, whileBLASTP can be used to align and compare the identity between amino acidsequences. When utilizing BLAST programs to calculate the percentidentity between a nucleic acid sequence encoding a therapeuticpolypeptide and another sequence, the default parameters of therespective programs can be used including the default for gap penalty.

Nucleic acids of the present invention may be detected by methods suchas Southern or Northern blot analysis (i.e., hybridization), PCR, or insitu hybridization analysis. Polypeptides are typically detected byimmunocytochemistry in transfected cell lines or by sodium dodecylsulphate (SDS)-polyacrylamide gel electrophoresis followed by CoomassieBlue-staining or Western blot analysis using antibodies (monoclonal orpolyclonal) that have specific binding affinity for the particularpolypeptide. Many of these methods are discussed in detail in Sambrooket al. (1989, Molecular Cloning: A Laboratory Manual, 2^(nd) Ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

Nucleic acid sequences encoding an immune co-stimulatory polypeptide andthe binding pair member can be operably linked to one another in aconstruct using conventional molecular biology techniques. See, forexample, Molecular Cloning: A Laboratory Manual (Sambrook et al., 2001,2^(nd) Ed., Cold Spring Harbor Laboratory Press) or Short Protocols inMolecular Biology (Ausubel et al., 2002, 5^(th) Ed., Current Protocols).Constructs suitable for use in these methods are commercially availableand used routinely in the art. Constructs can include elements necessaryfor expression such as promoter sequences, regulatory elements such asenhancer sequences, and response elements and/or inducible elements thatmodulate expression of a nucleic acid sequence. As used herein,“operably linked” refers to (i) positioning of a promoter and/or otherregulatory element(s) relative to a nucleic acid sequence in such a wayas to direct or regulate expression of the nucleic acid; and/or (ii)positioning the nucleic acid encoding the immune co-stimulatorypolypeptide and the nucleic acid encoding the binding pair member, suchthat the coding sequences are “in frame,” i.e., such that the constructencodes a fusion protein comprising the immune co-stimulatorypolypeptide and the binding pair member.

A construct can be propagated or expressed to generate a polypeptide ina host cell by methods known in the art. As used herein, the term “host”or “host cell” is meant to include not only prokaryotes, such as E.coli, but also eukaryotes, such as yeast, insect, plant and animalcells. Animal cells include, for example, COS cells and HeLa cells. Ahost cell can be transformed or transfected with a DNA molecule (e.g., aconstruct) using any of the techniques commonly known to those ofordinary skill in this art, such as calcium phosphate or lithium acetateprecipitation, electroporation, lipofection and particle bombardment.Host cells containing a vector of the present invention may be used forpurposes such as propagating the vector, producing a nucleic acid (e.g.,DNA or RNA), expressing an immune co-stimulatory polypeptide orfragments thereof, or expressing a fusion protein, as described above.

FIGS. 1A & 1B, 2A & 2B, 3A & 3B, 4A & 4B, 5A & 5B, and 7A & 7B showrepresentative nucleic acid sequences (SEQ ID NOs. 1, 3, 5, 7, 9 & 14)that include coding sequences for immune co-stimulatory moieties thatcomprise core streptavidin and an immune co-stimulatory polypeptide, andthe corresponding encoded amino acid sequences (SEQ ID NOs. 2, 4, 6, 8,10 & 15).

Immunotherapy

One embodiment of the present invention provides a method of generatingor enhancing an immune response against a first antigen or infectiousagent by administering to a patient in need thereof (a) a firstconjugate comprising (i) a conjugate member comprising a first immuneco-stimulatory polypeptide and (ii) a conjugate member comprising afirst member of a binding pair, and (b) a second conjugate comprising(i) a conjugate member comprising the first antigen or the infectiousagent and (ii) a conjugate member comprising a second member of thebinding pair. In an alternative embodiment, immune cells are treatedwith the first and second conjugates and then administered to thepatient. As discussed above, any immune co-stimulatory polypeptide andany antigen associated with a tumor or infectious agent (or theinfectious agent itself) may be used, as may any binding pair.

The present invention include the use of chimeric co-stimulatorymolecules with and without conjugation to the antigen of interest.

In embodiments where the conjugates are administered directly to thepatient, the first conjugate and the second conjugate can beadministered at substantially the same time or at different times. Inone embodiment, the conjugates are bound together via the bindingactivity of the binding pair members before administration to thepatient. For example, the first and second conjugates can be combined invitro and administered in a single composition. In another embodiment,the first conjugate is administered first, followed by administration ofthe second conjugate after a time sufficient for the immuneco-stimulatory polypeptide to bind to immune cells. This time period,for example, may vary from one to a few hours, to from one day to a fewdays, to from one week or longer.

The first and second conjugates can be administered to the patientsystemically or locally, such as by intravenous, intranasal, peritoneal,or subcutaneous injection. In one embodiment, one or more of thecomposition(s) are administered locally via direct injection into atumor site, such as by intratumoral injection, or into a site of localinfection. In another embodiment one or more of the compositions areadministered by different routes. For example, or one or morecompositions can be administered locally and one or more can beadministered systemically.

In embodiments where the conjugates are use to treat immune cells whichare then administered directly to patient, the immune cells can betreated with the first and the second conjugates at substantially thesame time or at different times. In one embodiment, the first and secondconjugates are bound together via the binding activity of the bindingpair members before being used to treat immune cells. For example, thefirst and second conjugates can be combined in a single composition andused to treat immune cells in vitro, such as by contacting immune cellswith the composition. In another embodiment, immune cells are treatedwith the first conjugate, followed after a period of time by treatmentwith the second conjugate. This time period, for example, may vary fromone to a few hours, to from one day to a few days, to from one week orlonger. The treated immune cells are administered to the patient by anymeans described above, including systemic or local administration, suchas intratumoral injection or injection into a site of local infection.

In accordance with one embodiment, the method further comprisesadministering a third conjugate or treating immune cells with a thirdconjugate. In one embodiment, the third conjugate comprises (i) aconjugate member comprising an immune co-stimulatory polypeptide and(ii) a conjugate member comprising a second antigen associated with thetumor or infectious agent or the infectious agent itself. The immuneco-stimulatory polypeptide of the third conjugate may be the same as ordifferent from the immune co-stimulatory polypeptide of the firstconjugate, and the second antigen may be the same as or different fromthe first antigen. In a specific aspect of this embodiment, the immuneco-stimulatory polypeptide and second antigen are bound together viainteractions of binding pair members associated with each of the immuneco-stimulatory polypeptide and second antigen. In accordance with thisembodiment, the first and second binding pair members of the thirdconjugate may be the same as or different from the first and secondbinding pair members of the first and second conjugates.

In one embodiment, the first conjugate comprises an immuneco-stimulatory polypeptide that binds to a constitutive receptor, suchas CD80, LIGHT, and CD40L, and the third conjugate comprises an immuneco-stimulatory polypeptide that binds to an inducible receptor, such as4-1BBL and OX40L.

Table 7 below provides a listing of costimulatory molecules that areconstitutive and inducible.

TABLE 7 Constitutive Inducible CD80-CSA CSA-4-1BBL CSA-CD40L CSA-OX40LCSA-LIGHT PD-L1-CSA GL50-CSA

Efficacy of cancer immunotherapy can be assessed by determining thedecrease in tumor cell proliferation and/or tumor size. The number oftumor cells is not static and reflects both the number of cellsundergoing cell division and the number of cells dying (e.g., byapoptosis). Increasing an individual's immune response against tumorcells may inhibit proliferation of the cells. Proliferation of tumorcells as used herein refers to an increase in the number of tumor cells(in vitro or in vivo) over a given period of time (e.g., hours, days,weeks, or months). Inhibiting the proliferation of tumor cells can bemeasured by a decrease in the rate of increase in tumor cell number, acomplete loss of tumor cells, or any decrease in proliferationtherebetween. A decrease in the size of a solid tumor is an indicationof an inhibition of proliferation of tumor cells.

The present invention offers an advantage over prior art cancer vaccinesby providing the ability to target TAAs specifically to DCs through theinteraction of the immune co-stimulatory polypeptide (such as 4-1BBL)with the its receptor. Moreover, the invention provides a vaccine thatcan be administered to a patient by injection and taken up by the DC invivo, leading to antigen presentation and activation without requiringthe isolation and ex vivo manipulation of DCs or the use of genetherapy.

Efficacy of immunotherapy against infection can be assessed bydetermining the patient's infection burden, and by assessing clinicalendpoints, such as fever or swelling.

The use of avidin/biotin binding pairs in accordance with the invention(or other mechanisms for providing higher order structures of immuneco-stimulatory molecules) offers the further advantage of providing atetrameric structure (or other multimeric structure) that permitscross-linking of the immune co-stimulatory receptor for a strongerresponse and that permits delivery of multiple antigen molecules to DCs.In one embodiment, the first binding pair member, i.e., the binding pairmember of the first conjugate (comprising the first immuneco-stimulatory polypeptide) is avidin, streptavidin or corestreptavidin, and the second binding pair member, i.e. the binding pairmember of the second conjugate (comprising the first antigen or theinfectious agent) is biotin. In another embodiment, the first bindingpair member is biotin and the second binding pair member is avidin,streptavidin, or core streptavidin.

The use of 4-1 BBL as the immune co-stimulatory polypeptide may offerfurther advantages, because stimulation of DCs with 4-1BBL has beenshown to nullify the suppressive function of T_(reg) cells that play adominant role in tumor evasion of the immune system. Thus, conjugates ofthe present invention comprising 4-1BBL and a TAA will deliver the TAAto DCs for effective presentation, activate DCs for the elaboration ofvarious cytokines, and nullify the function of T_(reg) cells whileboosting the function of Teff and NK cells for tumor eradication.

Modified Immune Cells

The invention also provides modified immune cells, and methods of makingthem, that are useful in immunotherapy methods as described above. Inaccordance with this aspect of the invention, there is provided a methodof modifying immune cells to generate or enhance an immune response to atumor expressing a first tumor associated antigen or to an infectiousagent. The method comprises contacting immune cells expressing areceptor for a first immune co-stimulatory polypeptide with (a) a firstconjugate comprising (i) a conjugate member comprising the first immuneco-stimulatory polypeptide and (ii) a conjugate member comprising afirst member of a binding pair; and (b) a second conjugate comprising(i) a conjugate member comprising an antigen associated with the tumoror infectious agent or the infectious agent and (ii) a conjugate membercomprising a second member of the binding pair. In accordance with thismethod, the first conjugate is conjugated to the immune cells viabinding between the immune co-stimulatory polypeptide and the receptor,and the second conjugate is conjugated to the immune cell via bindingbetween the first and second binding pair members.

The immune cells may be contacted with the first and second conjugatesby any means, and this may be effected in vivo or in vitro. For example,an in vivo method may comprise administering the first and secondconjugates to a patient comprising the immune cells and comprising or atrisk of comprising the tumor or infectious agent. In accordance withthis method, the first and second conjugates may be administeredsubstantially simultaneously (in the same or separate compositions) orsequentially (in separate compositions). In one embodiment where thepatient comprises a tumor, at least one of the first and secondconjugates is administered by intratumoral injection.

An exemplary in vitro method may comprise contacting immune cells withthe first and second conjugates in vitro, such as by contacting with asingle composition comprising the first and second conjugates, or bycontacting with first and second compositions comprising the first andsecond conjugates, respectively. When the conjugates are provided in asingle composition, they may be bound together via binding between thefirst and second binding pair members, as provided in the composition.

Any immune co-stimulatory polypeptide, antigen or infectious agent, andbinding pair members can be used in this aspect of the invention,including each described above.

Any immune cell expressing a receptor for the first immuneco-stimulatory polypeptide can be modified in accordance with thismethod. In one embodiment, the immune cells are T cells or neutrophils.Exemplary T cells include CD4+ cells, CD8+ cells, natural killer cells,monocytes and dendritic cells.

In a further embodiment of this aspect of the invention, the immunecells comprises a receptor for a second immune co-stimulatorypolypeptide, and the method further comprises contacting the immunecells with a third conjugate comprising (i) a first conjugate membercomprising the second immune co-stimulatory polypeptide and (ii) asecond conjugate member comprising a antigen associated with the tumoror infectious agent (or the infectious agent itself). In thisembodiment, the second immune co-stimulatory polypeptide may be the sameas or different from the first immune co-stimulatory polypeptide and thesecond antigen, if present, may be the same as or different from thefirst antigen, if present. In another specific embodiment, the first andsecond conjugate members further comprising first and second members ofa binding pair, respectively. In accordance with that embodiment, thefirst and second binding pair members of the third conjugate are thesame as or different from the first and second binding pair members ofthe first and second conjugates. Additionally, the first conjugatemember may be bound to the second conjugate member via binding betweenthe first and second binding pair members.

As with the first and second conjugates discussed above, the thirdconjugate may comprise any immune co-stimulatory polypeptide, antigen orinfectious agent and binding pair members, including any describedherein.

In a related aspect, the invention provides a population of immune cellsmade by this method. Such immune cells generate or enhance an immuneresponse to the tumor when contacted with other immune cells.

The invention also provides a modified immune cell expressing a receptorfor a first immune co-stimulatory polypeptide, wherein the modifiedimmune cell is modified with (a) a first conjugate comprising (i) aconjugate member comprising the first immune co-stimulatory polypeptideand (ii) a conjugate member comprising a first member of a binding pair;and (b) a second conjugate comprising (i) a conjugate member comprisinga first antigen or infectious agent and (ii) a conjugate membercomprising a second member of the binding pair. In accordance with thisembodiment, the first conjugate is conjugated to the immune cell viabinding between the immune co-stimulatory polypeptide and the receptor,and the second conjugate is conjugated to the immune cell via bindingbetween the first and second binding pair members.

As with the methods described above, any immune co-stimulatorypolypeptide, antigen or infectious agent, and binding pair members canbe used in this aspect of the invention, including each described above.

Further, any immune cell expressing a receptor for the first immuneco-stimulatory polypeptide can be modified in accordance with thismethod. In one embodiment, the immune cell is a T cell or neutrophil.Exemplary T cells include CD4+ cells, CD8+ cells, natural killer cells,monocytes and dendritic cells.

Immunostimulatory Moieties

The invention also provides immunostimulatory moieties that haveimmunostimulatory activity. The immunostimulatory moieties are usefulwhen administered alone, or when used as an adjuvant in conjunction withthe administration of an antigen or other immunostimulatory agent. Forexample, the immunostimulatory moieties are useful in the context ofvaccines, cancer immunotherapy, and the treatment of immune-baseddisorders. The immunostimulatory moieties can be formulated incompositions suitable for administration to an animal, and can beadministered to an animal in need of immunostimulation, such as ananimal receiving a vaccine, cancer immunotherapy, or undergoingtreatment for an immune-based disorder.

In accordance with one embodiment, the immunostimulatory moietycomprises any of the immune co-stimulatory polypeptides described above,such as 4-1BBL, CD86, ICOSL, PD-L1, PD-L2, B7-H3, B7-H4, OX40L, CD27L,CD30L, LIGHT, BAFF, APRIL, CD80 and CD40L. In another embodiment, theimmune co-stimulatory polypeptide is selected from the group consistingof 4-1BBL, ICOSL, PD-L1, PD-L2, OX40L, CD27L, CD30L, LIGHT, BAFF, andAPRIL. In yet another embodiment, the immune co-stimulatory polypeptideis 4-1BBL.

In accordance with one specific aspect of this embodiment, theimmunostimulatory moiety further comprises streptavidin or corestreptavidin. For example, the immunostimulatory moiety may be aconjugate or fusion protein comprising an immunostimulatory polypeptideand core streptavidin.

In another embodiment, the immunostimulatory moiety consists essentiallyof the immune co-stimulatory polypeptide and streptavidin or corestreptavidin. In accordance with this embodiment, the immunostimulatorymoiety does not comprise, and is not conjugated or otherwise bound toany other immunostimulatory agent, such as another immune co-stimulatorypolypeptide or antigen.

The invention also includes an immunostimulatory method that comprisesadministering an immunostimulatory moiety to a patient in need of immunestimulation. In one embodiment of this method, the immunostimulatorymoiety comprises an immune co-stimulatory polypeptide and streptavidinor core streptavidin. In another embodiment, the immunostimulatorymoiety consists essentially of the immune co-stimulatory polypeptide andstreptavidin or core streptavidin. In a further embodiment, the methodfurther comprises administering an antigen to the patient,simultaneously or sequentially (either before or after) administrationof the immunostimulatory moiety. In some embodiments of simultaneousadministration, the immunostimulatory moiety and antigen areadministered in a single composition, such as a mixture comprising theimmunostimulatory moiety and antigen. In other embodiments ofsimultaneous administration, the immunostimulatory moiety and antigenare administered in separate compositions. In some embodiments, theantigen is administered as an antigen-containing conjugate as describedabove, such as a conjugate comprising an antigen and a member of abinding pair. In other embodiments, the antigen is not administered as aconjugate comprising a member of a binding pair.

Another embodiment of the immunostimulatory method consists essentiallyof administering an immunostimulatory moiety that consists essentiallyof the immune co-stimulatory polypeptide and streptavidin or corestreptavidin. In accordance with this embodiment, no otherimmunostimulatory agent, such as another immune co-stimulatorypolypeptide or antigen, is administered that would become conjugated orotherwise bound to the immunostimulatory moiety. Thus, for example, nobiotinylated molecule, such as biotinylated cells or protein conjugatecomprising biotin, is administered.

While not wanting to be bound by any theory, it is believed that theimmunostimulatory moieties of the invention stimulate interactionsbetween cell surface immune receptors and their ligands, therebypromoting humoral and cellular immune responses. Immunostimulatorymoieties comprising streptavidin (or core streptavidin) form stabletetramers and oligomers that effectively engage receptors, and stimulateB cells, monocytes, and dendritic cells for the production of cytokines,chemokines, and up-regulation of immunostimulatory molecules.

The following examples illustrate the invention in more detail, and arenot intended to limit the scope of the invention in any respect.

EXAMPLES Experimental Methods

Animals. Adult inbred BALB/c (H-2^(d)) and C57BL/6 mice are purchasedfrom Jackson Laboratories (Bar Harbor, Me.). TCR transgenic OT-I,D011.10, C57BL/6.SJL animals are be purchased from Taconics (Germantown,N.Y.) and maintained under NIH Guidelines.

Establishment of A20 cells expressing OVA. An OVA construct was obtainedfrom Dr. Tom Mitchell of the University of Louisville and directionallycloned into the pcDNA3 vector (Invitrogen, San Diego, Calif.) restrictedwith BglII and EcoRI. After bacterial transformation and selection onampicillin medium, several clones were subjected to mini plasmidpreparation and digested with BglI/EcoRI to identify positive clones. Aclone containing the insert was then used for large plasmid preparationand transfection into A20 cells using Lipofectamine™ 2000 (Invitrogen)kit according to the manufacturer's instructions. Cells are be selectedin media containing G418 (Geneticin) and expression of OVA is determinedusing Western blots and antibodies against OVA or T cell proliferationassays.

Establishment of TC-1 transplantable cervical cancer model. A TC-1 tumormodel was established in C57BL/6 mice. The tumorigenic TC-1 cell linewas derived by co-transformation of primary C57BL/6 mouse lungepithelial cells with HPV-16 E6 and E7 and an activated ras oncogene,and has been characterized as a model for human cervical carcinoma. TC-1cells form tumors in syngeneic C57BL/6 mice. To establish the model,1×10⁵ tumor cells were transplanted into the right flank of C57BL/6 miceand animals were monitored for tumor growth.

Expression and purification of the recombinant 4-1BBL using insect DESexpression system. Stable transfectants expressing 4-1BBL using theDrosophila DES Expression System (Invitrogen; Carlsbad, Calif.) areestablished as described in Singh et al., 2003, Cancer Res. 63: 4067-73.Transfectants are induced for recombinant protein expression inDrosophila serum-free medium (Gibco; Carlsbad, Calif.) supplemented with1 mM copper sulfate for 72 hours in an incubator shaker set at 25° C.and 105 rpm. Culture supernatant is harvested by centrifugation andsubjected to large-scale purification using cobalt(ID-carboxymethylaspartate crosslinked agarose immobilized metalaffinity resin (BD-Talon, BD Biosciences) or Ni-NTA metal affinity resin(Qiagen), taking advantage of the 6×-His-tag engineered into theproteins.

Briefly, culture medium containing 4-1BBL are precipitated by dropwiseaddition of 95% ethanol to produce a final concentration of 10% ethanol.After an overnight incubation at 4° C. the precipitated 4-1BBL isredissolved in 1/10 of its starting volume with binding buffer (50 mMsodium phosphate pH 7.0; 500 mM sodium chloride; 0.5% Tween-20; 1%glycerol; 5 mM 2-mercaptoethanol). The metal affinity resin isequilibrated using 5× gel bed volume of binding buffer, added to theredissolved protein solution containing 4-1BBL, and incubated withend-over-end rotation for 45 minutes at room temperature. The 4-1BBLbound metal affinity resin is washed 2× with 50-100 ml of wash buffer(50 mM sodium phosphate pH 7.0; 500 mM sodium chloride). Bound 4-1BBL iseluted from the metal affinity resin with 2× gel bed volume of elutionbuffer (50 mM sodium phosphate pH 7.0; 500 mM sodium chloride 150 mMimidazole).

Purified 4-1BBL eluates are pooled and loaded into Amicon Ultra™(Millipore; Bedford, Mass.) centrifugal filter devices with 30 kDmolecular weight cut off membrane. The centrifugal filter devices arecentrifuged at 3000 rpm (2000×g) at 4° C. for 15 minutes. Sterile PBS isadded to the retentate and the filters are centrifuged again at 3000 rpm(2000×g). The retentate containing the concentrated/desalted 4-1BBL isaspirated from the centrifugal filter devices, placed in sterilecryovials, and stored in liquid nitrogen. The purity of the isolatedproteins is assessed by SDS-polyacrylamide gel electrophoresis. Proteinconcentration is determined using the BCA protein assay (Pierce)according to the manufacturer's instructions.

Expression and purification of biotinylated OVA. The OVA constructdescribed above is subcloned into the pAN and pAC vectors from Avidity,Inc. (Denver, Colo.) to express N-terminal as well as C-terminalAviTag-protein fusions, respectively. After bacterial transformation andselection on ampicillin medium, several clones are subjected to miniplasmid preparation and digested with the appropriate restrictionenzymes to identify positive clones. A clone with the insert is used forlarge plasmid preparation. Plasmids are used to transform AVB100 E.coli, a strain with the birA ligase gene stably integrated into thechromosome. Protein expression is induced with L-arabinose for highlevel of expression of OVA with the biotin tag. The expressed proteinsare purified using an AviTag antibody agarose. Purified OVA is assessedfor concentration, endotoxin level, and biotinylation using Western blotand alkaline phosphatase conjugated streptavidin for probing. Ifnecessary, endotoxin is removed using Detoxi-Gel Endotoxin Removing kit(Pierce). Biotinylated OVA is conjugated with a CSA-4-1BBL fusionprotein and tested in in vivo proliferation assays using OT-I TCRtransgenic cells, as discussed below. The protein is aliquoted andfrozen in −70° C. until use.

Proliferation assays. For in vivo proliferation assay, spleen and lymphnode cells are harvested from OT-I (OVA₂₅₇₋₂₆₄/K^(b)) TCR transgenicanimals. Cells are labeled with 5 μM CFSE (carboxyfluorescein diacetatesuccinimidylester) and one million CFSE-labeled cells are transferredinto CD45.1⁺ congeneic B6-SJL mice by tail-vein injection. After 24hours, animals are challenged with 10 μg OVA alone, OVA mixed with orconjugated to CSA or CSA-4-1BBL. Spleen and lymph nodes cells areharvested after 3 days and proliferation is determined by analysis ofCFSE dilution in CD8⁺ CD45.1⁻ (OT-1) cell populations in the lymphoidgate. Cells harvested from some animals that did not receive OVA proteinare used to determine the parent population for analysis.

In vitro proliferation assays are performed as follows: CFSE-labeledDO11.10 (OVA₃₂₃₋₃₃₉/I-A^(d)) TCR transgenic cells from BALB/c mice areused as responders against irradiated A20 transfectants expressing OVAat various ratios for 3 days. Cultures are harvested and analyzed inflow cytometry for proliferation.

Flow cytometry. Flow cytometric analysis is performed by first titratingthe primary and secondary antibodies of interest and then using theoptimum concentrations in flow cytometry using standard procedures. See,e.g., Mhoyan et al., 1997, Transplantation 64: 1665-70. Isotype-matchedantibodies serve as negative controls. Samples are run on a FACS Caliburor Vantage (Becton Dickinson; Mountain View, Calif.) and analysis isperformed using FlowJo software (TreeSoft).

Immunotherapy. Vaccinations are performed as follows. Briefly,CSA-4-1BBL fusion protein is mixed with biotinylated OVA in PBS atvarious molar ratios and then injected intraperitoneally into BALB/cmice for pre-vaccination, or for immunotherapy into animals that havebeen inoculated subcutaneously in the flank with a lethal dose of viableA20 (1×10⁶) cells. Controls include animals without vaccination or thosevaccinated with control proteins. Once detected, tumors are measuredevery other day using calipers and tumor size is reported as the averageof the longest diameter and the perpendicular diameter±standard error.Animals are euthanized when the tumor size reaches approximately 20 mmin diameter to avoid discomfort.

Statistics. The effect of treatments on tumor survival is estimatedusing Kaplan-Meier curves. The differences in survival between differentgroups is assessed using the log-rank test (generalized Savage/MantelCox). Procedures involving the comparison of data from groups ofindividual animals will first have the equality of variance examinedusing the F test (two groups) or Levene's test (multiple groups). Whenvariances are not equal, log transformations are performed. Whennormally distributed sample means are to be compared, the Student's ttest (two groups) or the Newman-Keuls test (multiple groups) is used.When the data is not normally distributed, the Mann-Whitney U test (twogroups) or the Kruskal-Wallis test (multiple groups) is used.Statistical significance is defined as P<0.05.

Example 1 CSA-4-1BBL Augments Alloantigen-Driven Responses

As discussed above, 4-1BBL plays an important role in the regulation ofadaptive and innate immune responses. 4-1BBL serves as a costimulatorymolecule for the activation of CD4⁺ and CD8⁺ T cells, NK cells, and DCsand inhibits the suppressive function of T_(reg) cells. Therefore, thismolecule can serve as a specific adjuvant for the generation of aneffective tumor response for cancer therapy.

The CSA-4-1BBL fusion protein forms tetrameric/oligomeric structure dueto the presence of the core streptavidin moiety, and is a solublemolecule. The immunostimulatory activity of CSA-4-1BBL on T cellresponses was demonstrated using allogeneic mixed lymphocytes reactions(MLR) as follows.

C57BL/6 mice lymph node cells were used as responders against BALB/cirradiated splenocytes in the presence or absence of CSA-4-1BBL.Cultures were labeled with [³H]thymidine for the last 18 hours of theculture period and proliferation was assessed. Cultures supplementedwith CSA-4-1BBL showed potent proliferative activity as compared withcontrols (FIG. 8).

Example 2 CSA-4-1BBL Enhances T cell Proliferation

To assess the relative activity of the 4-1-BBL fusion protein to amonoclonal antibody against 4-1BB, CD4⁺ and CD8⁺ T cells sorted by flowcytometry were polyclonally stimulated with a suboptimum concentrationof anti-CD3 antibody in the presence of various amounts of 4-1BBL fusionprotein and antibody in proliferation assays. The fusion protein had70-fold more activity on the proliferation of T cells than the antibody(FIG. 9).

Because this antibody Ab has been shown to have potent activity inanimal models of cancer immunotherapy, see, e.g., Melero et al., 1998,Cell Immunol. 190: 167-72; Melero et al., 1997 Nat. Med. 3: 682-85, thisdata indicates that the CSA-4-1BBL fusion protein will be a usefulcomponent of cancer vaccines, both as an adjuvant and as a vehicle todeliver TAAs to DCs.

Example 3 Effect Of A Biotinylated OVA/CSA-4-1-BBL Conjugate On CD8⁺ Tcells

Ovalbumin peptide (OVA) was biotinylated using a commercially availablekit (Pierce Biotechnology, Rockford, Ill.). Biotinylated OVA waspremixed in vitro with CSA-4-1BBL fusion protein for conjugation atvarious ratios and injected intraperitoneally into naïve C57BL/6.SJLanimals adoptively transferred with one million OT-1 T cells.Specifically, one million OT-I CD8⁺ T cells were labeled with CFSE andtransferred into B6.SJL mice that were immunized with biotinylatedovalbumin (10 μg/injection) (“OVA”) and CSA-4-1BBL (1 μg/injection)mixed with biotinylated OVA (“41BBL+OVA”) or conjugatedOVA-biotin/CSA-4-1BBL (“41BBL-OVA). (FIG. 10) The last panel of FIG. 10(“41BBL-OVA*”) shows the response for 5 μg of CSA-4-1BBL conjugated to10 μg biotinylated OVA. For controls, core streptavidin (“SA”) was usedat equimolar level as CSA-4-1BBL.

As shown in FIG. 10, 4-1BBL/OVA conjugates generated a potent (73.5%)proliferative response in OT-1 cells as compared with control “SA/OVA”conjugates (33.6%) or unconjugated, single proteins, “41BBL+OVA”(35.5%). The proliferative response was dose-dependent since a 5 μg doseof CSA-4-1BBL generated a much better response (94.5%) than a 1 μg dose(73.5%).

This example shows that the CSA-4-1BBL fusion protein increased theproliferative response of antigen-specific CD8⁺ T cells, indicating thatthe 4-1BBL-CSA/biotinylated antigen construct can successfully deliverantigen to professional APCs and activate these cells for the generationof an effective immune response.

Example 4 CSA-4-1BBL Delivers Antigens to DCs

This example demonstrates that CSA-41BBL effectively delivers antigen toDC. Biotinylated PE was used as a fluorescent antigen. Biotinylated PE(250 ng) was conjugated with 250 ng CSA-41BBL on ice for 30 min. Jaws IIDendritic cells (5×10⁵/well) were cultured for 16 hours withbiotinylated PE (250 ng/ml) or biotinylated PE/CSA-41BBL conjugate. Thelevel of PE was detected using flow cytometry. FIG. 11A is a histogramshowing the PE+ cells. The gray filled area represents untreated cells,the black dashed line represents cells treated with biotinylated PE, andthe black line represents cells treated with biotinylated PE/CSA-41BBLconjugate. FIG. 11B shows the mean fluorescence intensity (MFI) of PEfor each treatment, and demonstrates that the conjugate-treated cellsexhibited a significantly greater response.

Example 5 CSA-4-1BBL Activates DCs

This example demonstrates that 4-1BBL activates dendritic cells. Jaws IIDendritic cells (5×10⁵/well) were untreated or treated with 5 μg/mlCSA-41BBL conjugate or 5 μg/ml lipopolysaccharide (LPS) in the presenceof 5 ng/ml GM-CSF for 48 hours in 24-well plates. CD86 and MHC class IIlevels were analyzed using flow cytometry, as show in FIG. 12A. Thelight gray filled area represents isotype treated cells, the dark grayfilled are represents untreated cells, the black line representsCSA-4-1BBL treated cells, and the dashed line represents LPS treatedcells. FIG. 12B shows the mean fluorescence intensity (MFI) of CD86 andMHC class II, and demonstrates that the CSA-4-1BBL treated cellsexhibited a significantly greater response.

Example 6 CSA-4-1BBL Delivers Antigens to DCs and Activates DCs in vivo

This example demonstrates that CSA-4-1BBL delivers biotinylated antigensto dendritic cells and drive these cells to activation in vivo.Biotinylated OVA was contacted with CSA-41BBL to yield a biotinylatedOVA/CSA-4-1BBL conjugate. That conjugate or a biotinylated OVA/CSAconjugate was injected intravenously into naïve C57BL/6 mice. 24 hourslater, animals were euthanized and spleen cells were harvested.Dendritic cell activation was analyzed using flow cytometry in CD11c+cell populations. The mean florescence intensity (MFI) of CD40, CD86 andMHC class II expression on dendritic cells from naïve, biotinylatedOVA-SA treated, and biotinylated OVA/CSA-41-BBL treated animals weredetermined, as shown in FIG. 13. This figure demonstrates that thebiotinylated OVA/CSA-41-BBL treated animals exhibited a significantlygreater response.

Example 7 CSA-4-1BBL Neutralizes The Suppressive Function Of Treg Cells

As discussed above, naturally occurring CD4⁺CD25⁺FoxP3⁺ Treg cellsconstitutively express the 4-1BB receptor and, as such, respond to4-1BBL stimulation. The following example demonstrates the stimulatoryactivity of the 4-1BBL fusion protein on Treg cells.

CD4⁺ CD25⁻Teff cells and CD4⁺ CD25⁺ Treg cells were isolated using flowsorting, cultured in a 1:1 ratio in the presence of irradiated syngeneiccells and anti-CD3 antibody. To differentiate between the proliferationof CD4⁺CD25⁺ (DP) versus CD4⁺CD25⁻ (SP) T cells in co-cultureexperiments, CD4⁺CD25⁻ T cells were stained with carboxyfluoresceindiacetate succinimidylester (CFSE, Molecular Probes, OR) and used insuppression assays. Briefly, cells were washed with PBS, incubated in 4ml of 2.5 μM CFSE/1×10⁶ cells (ratio was kept when lower amount of cellswere labeled) for 7 min at room temperature. Cells were then incubatedin two volumes of fetal bovine serum for 1 min, and washed 2 times withPBS to ensure removal of all excess CFSE. Proliferation was assessedusing flow cytometry.

Treg cells did not respond to anti-CD3 stimulation as they are anergic,but showed moderate proliferation in response to 4-1BBL (FIG. 15).Notably, Treg cells inhibited the proliferative response of Teff cells,an effect that could be reversed by the addition of 4-1BBL. This isconsistent with data using naïve Treg cells, where the suppressiveeffect of expanded cells was neutralized by the presence of CSA-4-1BBL(see below).

These data confirm the immunomodulatory effects of 4-1BBL, and itsutility for cancer immunotherapy. For example, the 4-1BBL fusion proteinboosts Teff functions while downregulating the inhibitory function ofTreg cells for a more robust anti-tumor immune response.

Example 8 Dual Role of 4-1BBL

The role of 4-1BB/4-1BBL-mediated signaling in the regulation of Tregfunction has been the subject of two recent studies with opposingfindings. While one study demonstrated that 4-1BB signaling neutralizesthe suppressive function of Treg cells, Choi et al., 2004, J. Leukoc.Biol. 75: 785-91, the other reported that 4-1BB signaling mediates Tregproliferation without a major effect on their suppressive function,Zheng et al., 2004, J. Immunol. 173: 2428-34. To clarify thisdiscrepancy, the role of 4-1BB signaling in Treg function wasinvestigated using a CSA-4-1BBL fusion protein.

CD4⁺CD25⁻ (single positive; SP) and CD4⁺CD25⁺ (double positive; DP) Tcells were sorted from the spleen and peripheral lymph nodes of naïveBALB/c mice and cultured alone or at 1:1 ratio for 3 days. Cultures weresupplemented with irradiated splenocytes, an anti-CD3 antibody (0.5μg/ml), and the concentrations (μg/ml) of 4-1BBL or equimolar amounts ofcontrol CSA protein indicated in FIG. 14A. CD4⁺CD25⁺ double positive(DP) T cells purified from naïve BALB/c mice using flow sorting markedlyinhibited the proliferative response of single positive (SP) CD4⁺CD25⁻Teff cells induced by an antibody against CD3 in co-culture experiments.This suppressive effect was effectively and specifically reversed bysupplementing cultures with 1 μg/ml CSA-4-1BBL, but not control CSA usedat an equimolar level.

To test whether the observed reversal of suppression by the CSA-4-1BBLfusion protein is due to the restoration of the proliferative responseof SP cells, SP cells were labeled with CFSE and used in co-cultureexperiments in the presence of CSA-4-1BBL (0.5 μg/ml) or CSA as acontrol protein. The CSA-4-1BBL increased the proliferation of SP cellsfrom 44% for control and 46% for CSA protein to 60%. DP cellssignificantly reduced the proliferation of SP T cells (16%), which wassignificantly restored (34%) by 4-1BBL, but not CSA control protein(17%). (FIG. 14B) These data demonstrate that the 4-1BBL fusion proteindown-regulates the suppressive function of Treg cells.

Thus, our work shows that the CSA-4-1BBL fusion protein manifested twoopposing activities on Treg cells. One the one hand, it synergized withanti-CD3 antibodies and IL-2 to promote Treg cell expansion. On theother hand, it blocked the suppressive function of both naïve andactivated Treg cells, but only when the Treg cells were in contact withthe 4-1BBL fusion protein, since its removal from the culture mediumresulted in recovery of the suppressive function.

This latter effect of 4-1BBL may have some significance in the contextof tumors and infections that use Treg cells as immune evasionmechanisms.

Example 9 Use of Antigen-4-1-BBL Conjugate As Cancer Vaccine (a)Production of A20 Transfectants Expressing OVA as a Soluble Protein.

The OVA construct described above are transfected into A20 cells usingLipofectamine™ 2000 kit according to the manufacturer's (Invitrogen)protocol. Stable transfectants are selected in G418 selection medium,cloned at single cell level, and tested for the expression of OVA usingWestern blots. Clones with significant level of OVA expression are asstimulators for DO11.10 CD4⁺ T cells specific for OVA peptide in CFSEproliferation assays as described with reference to FIG. 10 above. Onceconfirmed positive for OVA expression, one million live A20 cells areinjected subcutaneously into the right flank of BALB/c mice. Animals aremonitored for tumor development and survival every other day, andeuthanized when tumors reach a size of 20 mm in diameter. Animalsinoculated with the parental A20 cells will serve as control for tumorgrowth. A20 cell transfectants expressing OVA will form tumors wheninjected into syngeneic BALB/c mice.

(b) Production of CSA-4-1BBL Fusion Protein Using Insect DES System.

A CSA-4-1BBL protein is made using the DES expression system(Invitrogen) and our established protocols. See, e.g., Singh et al.,2003, Cancer Res. 63: 4067-73; Yolcu et al., 2002, Immunity 17: 795-808.The fusion protein is purified using immobilized metal based affinitychromatography taking advantage of the 6×His tag engineered into theCSA-4-1BBL fusion protein. The protein is desalted, concentrated byultrafiltration, and analyzed by SDS-PAGE for purity. Proteinpreparations are assessed for concentration using the bicinchoninic acid(BCA) assay (Pierce) and tested for the presence of endotoxin usingQCL-1000® Chromogenic LAL endpoint assay from Cambrex.

(c) Biotinylation of OVA

Maleimide activated, endotoxin-free, chicken OVA (Pierce) isbiotinylated using the DSB-X Biotin Labeling Kit according to themanufacturer's (Molecular Probes, San Diego, Calif.) protocol. Followingextensive dialysis in PBS, biotinylated OVA is assessed forconcentration, endotoxin level, and biotinylation using Western blot andalkaline phosphatase conjugated streptavidin for probing. If necessary,endotoxin will is removed using Detoxi-Gel Endotoxin Removing kit(Pierce). Biotinylated OVA is conjugated with the CSA-4-1BBL fusionprotein. The protein conjugate is aliquoted and frozen at −80° C. untiluse.

(d) Use of Biotinylated OVA/CSA-4-1-BBL Conjugate As Cancer Vaccine

CSA-4-1BBL and biotinylated OVA are premixed in PBS at various molarratios, such as 1:1, 1:5, 1:10, 5:1, and 10:1 4-1BBL:OVA, and injectedintraperitoneally into a group of BALB/c mice at various doses (such as10, 50, and 100 ug of OVA) at three weekly intervals. Animals injectedwith streptavidin conjugated to biotinylated OVA, biotinylated OVAalone, or unbiotinylated OVA mixed with CSA-4-1BBL will serve ascontrols.

Animals are challenged subcutaneously with 1 million live A20 tumorcells in the right flank, monitored for tumor development and survivalevery other day, and euthanized when tumors reach a size of 20 mm indiameter.

Vaccination with the biotinylated OVA/CSA-4-1-BBL conjugate willgenerate a potent anti-tumor immune response, leading to the preventionof tumor growth. Vaccination with unconjugated 4-1BBL and OVA may alsogenerate a response, but any such response will be smaller than thatgenerated by the antigen/4-1-BBL conjugate. Vaccination with OVA aloneor CSA-OVA may only produce minimal responses, and as such should beineffective in preventing tumor growth.

Example 10 Early/Late Vaccination With Antigen-4-1-BBL Conjugate

As discussed above, tumors evade the immune system by various mechanismsdeveloped over the course of tumor growth. The efficacy of theconjugates of the present invention early in tumor progression isdemonstrated by vaccinating animals concurrently with tumor challenge,when immune evasion mechanisms have not been established. The efficacyof the conjugates of the present invention against established tumors isdemonstrated by vaccinating animals once tumors have been established,and have fully developed immune evasion mechanisms.

(a) Efficacy Early In Tumor Progression

BALB/c mice are challenged with one million live A20 cells on the rightflank and simultaneously vaccinated intraperitoneally with biotinylatedOVA/CSA-4-1BBL conjugate. Vaccination with the antigen/4-1BBL conjugateis repeated once a week for four weeks, by which time the tumor incontrol animals will reach a size of 10-15 mm in diameter. Unmanipulatedanimals and animals vaccinated with CSA-OVA conjugates will serve ascontrols.

(b) Efficacy Against Established Tumors

BALB/c mice are inoculated subcutaneously in the right flank with 1million live A20 tumor cells. Animals are monitored for tumordevelopment and vaccinated with biotinylated OVA/CSA-4-1BBL conjugatewhen tumors reach a size of 4-6 mm in diameter. The vaccination protocolwill initially involve weekly intraperitoneal. injections until thetumor either disappears or reaches a size of 20 mm in diameter.

Animals that effectively eradicate their tumors will be challenged with2 million live A20 cells 60 days after tumor disappearance to test thememory response.

While not wanting to be bound by any theory, the antigen/4-1BBLconjugate of the invention may have greater efficacy in preventing thegrowth of tumor when administered early in tumor progression, ascompared with administration once tumors are established, due to thelack of various suppressive mechanisms early in the course of tumorprogression. Nevertheless, the antigen/4-1BBL conjugate will showefficacy in eradicating established tumors due to the specific targetingof antigen to DCs for efficient antigen presentation, activation of DCsfor the generation of a danger signal (adjuvant effect), anddownregulation of Treg cells' suppressive functions. In addition to theindirect effect on DCs, repeated injection with the vaccine may furtherboost the immune system by engaging 4-1BB receptor on activated T and NKcells, leading to their vigorous proliferation, survival, and memory Tcell function.

Example 11 Efficacy of Antigen-4-1-BBL Conjugate By Bystander Effect

The following example will demonstrate that the biotinylatedOVA/CSA-4-1BBL conjugate generates an immune responses against undefinedA20 tumor antigens (other than OVA), either by bystander effect orepitope spreading.

BALB/c animals are inoculated with A20 expressing OVA in the right flankand parental unmodified A20 cells in the left flank. Once tumors arepalpable, animals are vaccinated with the biotinylated OVA/CSA-4-1BBLconjugate. The vaccination schedule outlined above is followed, but maybe modified as needed to enhance efficacy. Animals are monitored for thegrowth of both tumor types.

Alternatively, animals having successfully eradicated their tumorfollowing vaccination with biotinylated OVA/CSA-4-1BBL conjugate (suchas in the example above) are challenged subcutaneously with 2 millionparental A20 cells on the opposite flank 60 days after the eradicationof A20 tumors expressing OVA.

The biotinylated OVA/CSA-4-1BBL conjugate vaccine will show efficacyagainst parental A20 tumors that lack OVA as a TAA. For example, aneffective immune response against OVA will lead to the killing of thetumor, shedding of tumor antigens, and capture and presentation by APCsfor the generation of T cell responses against a new set of TAAs. Theeradication of parental tumors may further be facilitated by bystandereffects generated against A20-OVA tumors.

Example 12 Production Of Biotinylated Antigen Using Bacterial ExpressionSystem

In some circumstances, it may be advantageous to produce geneticallybiotinylated antigens for used as the antigenic component of the vaccineof the present invention. The Biotin AviTag technology of Avidity, Inc.(Denver, Colo.) may be used in this regard. The Biotin AviTag iscomprised of a unique 15 amino acid peptide that is recognized by biotinligase, BirA, that attaches biotin to the lysine residue in the peptidesequence. The Biotin AviTag can be genetically fused to any protein ofinterest, allowing the protein to be tagged with a biotin molecule.

cDNA encoding OVA is subcloned into the pAN and pAC vector to expressN-terminal as well as C-terminal AviTag-protein fusions, respectively.AVB100 E. coli B strain with a birA gene stably integrated into thechromosome is transformed and induced with L-arabinose for high level ofexpression of OVA carrying a biotin tag. The expressed proteins arepurified using an AviTag antibody agarose. Purified OVA is assessed forconcentration, endotoxin level, and biotinylation using BCA kit,QCL-1000® Chromogenic LAL kit, and Western blots probed with alkalinephosphatase conjugated streptavidin. If necessary, endotoxin is removedusing Detoxi-Gel Endotoxin Removing kit (Pierce).

Biotinylated OVA is conjugated with CSA-4-1BBL as described above. Theprotein conjugate is aliquoted and frozen at −80° C. until use.

Example 13 Use of Antigen/4-1BBL Conjugates Comprising TERT or Survivin

The biotinylated antigen/CSA-4-1BBL conjugate exemplified above withbiotinylated OVA/CSA-4-1BBL can be used in any vaccine setting with anyantigen. In the context of cancer vaccines, two universal human TAAs,telomerase reverse transcriptase and survivin, may be advantageousantigenic components of a biotinylated antigen/CSA-4-1BBL conjugate ofthe present invention.

Example 14 E7/4-1BBL Conjugates

As discussed above, a conjugate of the present invention comprising thehuman papillomavirus E7 antigen as the antigen component is usefulagainst cervical cancer. This example relates to this specificembodiment of the invention.

(a) Production of Biotinylated HPV-16 E7

Biotinylated E7 is used as the antigenic component of a conjugateaccording to the present invention useful as an HPV vaccine. In oneembodiment, full-length E7 protein is to provide a maximum number ofepitopes. A cDNA encoding full-length HPV-16 E7 is cloned by RT-PCRusing total RNA from TC-1 cells. After sequence verification, the cDNAis subcloned into the pMIB/V5-His vector (Invitrogen) in frame with the6×-His tag for constitutive expression and secretion in the DES system.Secreted protein is purified using a metal affinity resin as describedabove. Purified E7 is biotinylated in vitro using EZ-LinkSulfo-NHS-LC-Biotin following the manufacturer's protocol (Pierce).Briefly, purified, concentrated E7 is buffer-exchanged in phosphatebuffered saline (PBS) and incubated with EZ-Link Sulfo-NHS-LC-Biotin atroom temperature for 1 hour. Unconjugated biotin is removed usingtangential flow filtration (Spectrum Labs, NJ).

(b) Production of E7/4-1BBL Conjugates

A conjugate comprising E7 and 4-1BBL is produced using biotinylated E7and a CSA-4-1BBL fusion protein, following the general proceduresdescribed above. For comparison, an E7/4-1BBL fusion protein is producedas follows. cDNA encoding E7 and 4-1BBL is subcloned into thepMIB/V5-His vector (Invitrogen) in frame with the 6×-His tag forconstitutive expression and secretion in the DES system, and the proteinis expressed and purified as described above.

(c) Binding Activity of E7/4-1BBL Conjugate

The biotin binding and 4-1BB receptor binding activity of the E7/4-1BBLconjugate is assessed as follows.

For biotin binding, TC-1 cells are biotinylated and incubated withCSA-4-1BBL (100 ng/10⁶ cells) in PBS on ice. Cells are extensivelywashed with PBS, stained with a fluorochrome-labeled antibody against4-1BBL, and analyzed using flow cytometry. Biotinylated cells conjugatedwith CSA serve as controls.

To test binding of the conjugate or fusion protein to 4-1BB receptor onactivated T cells, splenocytes from C57BL/B6 mice are activated with 5μg/ml of concanavalin A (Con A) for 36 hrs, washed with PBS andincubated with various concentrations of the conjugate or fusion proteinon ice. Cells are washed extensively and stained with the appropriatefluorochrome-labeled antibodies to 4-1BBL, core streptavidin, or E7, andanalyzed in flow cytometry.

Binding of CSA-4-1BBL conjugate to biotinylated E7 is determined byfirst forming conjugates using the proteins in a 1:4 ratio(CSA-4-1BBL:E7), following the stoichiometry of CSA-biotin binding, andthen testing the conjugates in a sandwich ELISA. Briefly, the conjugatedproteins are bound to 96-well plates coated with anti-E7 antibody,washed, and then incubated with a reactive anti-streptavidin antibody tomeasure the amount of E7/4-1BBL complex present. After confirmingformation of conjugates, they are assessed for the ability to bind to4-1BB receptor on activated T cells as described above.

Example 15 Immune Responses Induced By E7/4-1BBL Conjugate (a)Optimization of Dose.

Optimum doses of a vaccine comprising an E7/4-1-BBL conjugate may beassessed as follows. A conjugate comprising biotinylated E7 andCSA-4-1BBL is formed by mixing biotinylated E7 and CSA-4-1BBL at tworatios (such as CSA-4-1BBL:E7 of 1:4 and 1:8) using 1, 10 or 50 μgbiotinylated E7. Comparable amounts of control unbiotinylated E7 alsoare used. (These doses of E7 are based on studies demonstrating thatvaccination with 50 μg of E7 is effective to generate a protectiveimmune response against TC-1 cells.) Optimum ratios of 4-1BBL:E7 andoptimum amounts of antigen can be determined and adjustedexperimentally, by assessing immune responses to the vaccine undervarious protocols, such as those described below.

(b) Tetramer Analysis

Tetramer staining permits assessment of vaccine efficacy with regard tothe expansion of CD8⁺ T cells. C57BL/6 female mice are injectedintraperitoneally with the above-described vaccine preparations in PBS.Mice injected with PBS, CSA-4-1BBL, CSA-4-1BBL+unbiotinylated E7, orE7-4-1BBL fusion protein serve as controls. A second equivalent dose isgiven intraperitoneally 10 days later, and three days after the lastvaccination, splenocytes are harvested and the number of E7-specificCD8+ T cells are quantitated using tetramer technology and flowcytometry. Briefly, splenocytes from immunized animals are labeled withFITC-anti-CD8 antibody and PE-tetramers of MHC class I H-2D^(b)molecules loaded with the immunodominant epitope of E7, peptide 49-57(RAHYNIVTF). (The tetramer can be obtained from the National Institutesof Health Tetramer Facility (Atlanta, Ga.)). Class I H-2D^(b) moleculesloaded with Sendai virus nucleoprotein 324-332 peptide (FAPGNYPAL)serves as a negative control. After staining, cells are analyzed by flowcytometry to quantify the percentage of CD8+ T cells positive for thetetramer.

(c) Intracellular IFN-γ Analysis.

The characterization of vaccine-induced CD8+ T cells for the expressionof IFN-γ, a signature cytokine for effector CD8⁺ T cells, permitsassessment of the function of the T cells. Female C57BL/6 mice areinjected intraperitoneally with an optimum dose of biotinylatedE7/CSA-4-1BBL conjugate vaccine (determined as described above), and 10days later splenocytes from immunized animals are harvested andcocultured with irradiated TC-1 cells expressing E7 for 5 days, and thensupplemented with the Golgi transport inhibitor brefeldin A overnight.Live cells are harvested using Ficoll gradients and incubated withanti-mouse Fcγ receptor antibody (2.4G2 from American Type CultureCollection) for 1 hour followed by staining with FITC-labeled anti-CD8antibody. Cells are then fixed, permeabilized, stained for PE-labeledanti-IFN-γ antibody (Pharmingen) and analyzed by flow cytometry. Cellsstained with isotype antibodies serve as controls. Splenocytes fromanimals immunized with PBS, E7-4-1BBL fusion protein, orCSa-4-1BBL+unbiotinylated E7 serve as controls.

(d) Killing Response

The ability of vaccine-induced CD8⁺ T cells to lyse TC-1 cellsexpressing E7 molecule is assessed as follows. Splenocytes harvestedfrom animals vaccinated as described above are cocultured in thepresence of 100 μg/ml E7 protein for 5 days. Cultures are supplementedwith 50 U/ml exogenous IL-2 to support the growth of CD8+ T cells.Viable splenocytes are recovered using Ficoll gradients and used aseffector cells against TC-1 target cells at various effector:targetratios (such as 1:1, 10:1, 20:1, 40:1, and 80:1) in the JAM assay. See,e.g., Singh et al., 2003, Cancer Res. 63: 4067-73. Because directkilling of tumor cells by CD8⁺ T cells is important to cancerimmunotherapy, demonstration of efficacy in this assay will furthersupport the efficacy of the vaccine against cervical cancer.

(e) CD4+ T Cell Proliferation Response

The efficacy of biotinylated E7/CSA-4-1BBL in the induction of a CD4+ Tcell response is assessed as follows. Splenocytes from immunized animalsare labeled with CFSE and cocultured with recombinant E7 protein underthe same culture conditions as described above, except that IL-2 is notbe added to the cultures. Cells are harvested at various days duringculturing, stained with an APC-CD4 antibody, and analyzed forproliferation using flow cytometry. Cultures without E7 protein or withOVA protein serve as controls. Because there is a general consensus thata CD4+ T cell response is important for CD8+ T cell and B cellresponses, demonstration of efficacy in this assay will further supportthe efficacy of the vaccine against cervical cancer.

(f) Humoral Response

The ability of in vivo treatment with a biotinylated E7/CSA-4-1BBLvaccine to generate a humoral response is assessed as follows. Bloodfrom vaccinated mice is collected, and serum is isolated and used toscreen Maxisorb ELISA plates (Nalgene Nunc International) coated with E7protein. Anti-E7 IgG and IgM is detected with horseradishperoxidase-conjugated goat anti-mouse IgG and goat anti-mouse IgMantibodies. Controls include sera harvested from animals immunized withPBS and control proteins such as those described above.

The biotinylated E7/CSA-4-1BBL conjugate vaccine will generate potentresponses in these assays. Vaccination with E7-4-1BBL fusion protein mayalso generate a response, but any such response is expected to be ofsmaller magnitude. Vaccination with CSA-4-1BBL plus unbiotinylated E7may produce a response, but any such response will not be as strong asthat of the conjugate vaccine because the uptake of E7 antigen by APCswill be a random event, and not as efficient as the targeted delivery ofE7 to APCs achieved by the conjugate.

Example 16 Therapeutic Efficacy of E7/4-1BBL Conjugate

The efficacy of a vaccine comprising an E7/4-1BBL conjugate of thepresent invention in preventing and eradicating tumor formation in TC-1transplantable tumor models is assessed in two different settings. Thefirst setting involves vaccination prior to tumor injections, when theimmune evasion mechanisms have not yet developed. The second settinginvolves vaccination against established tumors with fully developedimmune evasion mechanisms. C57BL/6 mice are injected with TC-1 cells toinduce tumor formation, and vaccinated pre- and post-TC-1 injection withbiotinylated E7/CSA-4-1BBL conjugate. Immune responses are assessed asdescribed above. Mice also are monitored for tumor development andsurvival every other day, and euthanized when tumors reach a size of 20mm in diameter.

(a) Efficacy of E7/4-1BBL Conjugate Against Subsequent Tumor Challenge.

The following example will demonstate that immunization with anE7/4-1BBL conjugate of the present invention generates protectiveimmunity against a subsequent tumor challenge.

Female C57BL/6 mice are injected intraperitoneally with PBS alone,CSA-4-1BBL alone, CSA-4-1BBL mixed with unbiotinylated E7, abiotinylated E7/CSA-4-1BBL conjugate of the invention, and a E-7-4-1BBLfusion protein. Doses optimized as described above are used. A secondequivalent dose is given subcutaneously 14 days after the first dose.TC-1 cells are harvested, resuspended in sterile PBS, and used forinjection fourteen days after the last immunization. Mice are challengedsubcutaneously in the right flank with 1×10⁵ TC-1 cells (day 0) andobserved for 60 days. As a control to verify the specificity of theconjugate for E7⁺ tumors, one set of immunized mice are challenged withA20 cancer cells. Mice are monitored for tumor development and survivalevery other day, and euthanized when tumors reach a size of 20 mm indiameter. Animals that do not develop tumors are re-challenged with1×10⁶ TC-1 cells 60 days after the first tumor challenge to test thememory response. At fourteen day intervals, mice from each group aresacrificed and their splenocytes are harvested. Splenocytes are used todetermine CD8+ T cell responses using tetramer staining and cytokinestaining as described above.

(b) Efficacy of E7/4-1BBL Conjugate Against Existing Tumors

The therapeutic effects of an E7/4-1BBL conjugate of the presentinvention against pre-existing tumors is demonstrated as follows.

Female C57BL/6 mice are injected subcutaneously on the right flank withTC-1 cells and vaccinated when 100% of the mice have palpable tumors.Vaccines are administered doses optimized as described aboveintraperitoneally every week until the tumor size reaches 20 mm indiameter, at which time the mice are euthanized. The growth rate of thetumors and morbidity is assessed for 60 days. In addition, long-termsurvival is assessed and followed over 90 days.

Vaccination with a biotinylated E7/CSA-4-1BBL conjugate of the inventionwill generate a potent anti-tumor immune response in both settings,leading to the prevention of tumor growth and the eradication ofexisting tumors. Vaccination with unconjugated CSA-4-1BBL and E7 andwith the E7-4-1BBL fusion protein may also generate an anti-tumorresponse, but any such response will be minimal and likely ineffectivein preventing tumor growth or eradicating existing tumors.

Example 17 Conjugates Comprising Influenza A Antigens

cDNAs of influenza proteins of interest (e.g., H1, N1, NP and/or MP2)are generated by the reverse transcriptase-polymerase chain reactionfrom influenza A RNA. The cDNAs are subcloned into the pCSA vector, andtransfected into Drosophila insect cells for the establishment of stabletransfectants.

Taking advantage of a 6×-His tag engineered into the proteins, thesecreted H1, N1, NP and MP2 proteins are purified from the Drosophilaculture media using a metal affinity resin and tangential flowfiltration (methods and techniques already employed by ApoImmune).Purified proteins are analyzed by gel electrophoresis, immunoblottechniques, matrix assisted laser desorption/ionization—massspectrometry (MALDI-MS), and analytical ultracentrifugation.

CSA-4-1BBL (made as described above) is mixed at a molar ratio of 1:4with biotinylated H1, N1, NP or MP2 to form an Influenza A antigen/4-1BBL conjugate. Briefly, biotinylated H1, N1, NP or MP2 is incubated withCSA-4-1BBL for one hour at 4 C. Unbiotinylated H1, N1, NP or MP2 areincubated with CSA-4-1BBL to serve as unconjugated controls. Theconjugates can be formulated into compositions useful as vaccines.

Example 18 Vaccination Against Influenza A (a) Dosing Optimization

C57BL/6 mice are vaccinated with varying doses of the Influenza Aantigen/4-1BBL conjugates described above. Immune responses in the miceare determined using standard immunological techniques includingtetramer technology, cytokine staining, cytotoxity assays, and themeasurement of humoral responses, as described above. Initial resultsare used to determine an optimal dosing regimen for the vaccines.

(b) Vaccination With Infection Challenge

The protective and therapeutic efficacy of the Influenza Aantigen/4-1BBL conjugate vaccines is demonstrated in mice challengedwith influenza A as follows. Human influenza virus-infected animals aretreated with Influenza A antigen/4-1BBL conjugate vaccines pre- andpost-infection and viral titers are measured to determine the efficacyof treatment. Lungs from vaccinated and control infected animals areharvested days 1, 3, 5, 7, and 9 post-infection. Weight loss isdetermined daily as an indirect measurement of morbidity.

In another series of experiments, lung pathology is evaluated andpulmonary viral titers are determined. For this purpose, lungs arehomogenized and viral supernatants are collected followingcentrifugation of the homogenate at 1500×g for 15 min and frozen at −80°C. until subsequent analysis. Dilutions of viral supernatants from lungsare added to 3×10⁴ MDCK cells/well in a 96-well U-bottom plate for 24hours at 37° C., media is removed from wells and serum-free media isadded. Four days later, virus titers are determined by using standardcurve of known virus concentration and the Reed-Munch calculation ofTCID after identifying the dilution at which the culture supernatants nolonger agglutinate chicken red blood cells.

Example 19 Immune Co-stimulatory CD40L Moiety

The human monocytic leukemia THP-1 and mouse A20 B-cell lymphoma linesused in this example were purchased from the American Type CultureCollection (ATCC, Rockville, Md., USA). A20 cells were cultured in DMEM(GIBCO, Gaithersburg, Md., USA) supplemented with 10% heat-inactivatedfetal bovine serum (FBS; Valley Biomedical, Winchester, Va., USA), 12 mML-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin (all fromGIBCO) and 50 μM 2-mercaptoethanol (Sigma, St. Louis, Mo., USA). THP-1cells were cultured in RPMI supplemented with 5% FBS, 100 U/mlpenicillin and 0.1 mM Hepes buffer (GIBCO) at 37° C. in a humidified 5%CO₂ incubator. Cells were grown in suspension at 37° C. in 5% CO₂.

The Chinese Hamster ovary (CHO) and stable mouse CD154 transfectedCHO(CHO-mCD40L) lines used in this example were provided by Dr. GailBishop (University of Iowa) and were maintained in RPMI 1640 (GIBCO)containing 100 mM Hepes, 50 μg/ml gentamicin and 5% FBS.

Primary monocytes isolated by counterflow elutriation from humanperipheral blood mononuclear cells were a gift from Dr. Larry Wahl(NICDR).

Immortalized macrophage cell lines from CD40 knock out mice (CD40KO cellline) were established by infecting bone marrow cells with the murinerecombinant J2 retrovirus containing the v-myc and v-raf oncogenes aspreviously described. See, e.g., Clemon-Miller et al., 2000, Immunobiol.202: 477-92.

For the generation of stable human CD40 expressing transfectants, the J2transformed lines were electroporated with 10 μg of DNA at 600 v, 20μsecs, and 2 pulses. Zeocin (100 μg/ml) was added to the medium 24 hrafter transfection and resistant colonies were stained for surfaceexpression of CD40. High CD40 expressing cells were sorted using theFACS Vantage SE (Becton Dickinson, San Jose Calif., USA) and maintainedfor use in these studies.

(a) Cloning and Expression of CSA-CD40L Moieties

The gene encoding CSA was cloned using genomic DNA isolated fromStreptomyces avidinii as a template and specific primers in PCR (a and bin FIG. 16). The extracellular domain of human CD40L was cloned usingthe first strand cDNA generated from total RNA isolated fromphytohaemagglutinin (PHA) activated human peripheral blood lymphocytesas a template and CD40L-specific primers (c and d in FIG. 16) in PCR.The murine CD40L was cloned in the same manner as CSA-hCD40L using totalRNA isolated from mouse splenocytes activated with concanavalin A(ConA).

The CSA/CD40L gene was then subcloned in frame into the pMT/BiP/V5-HisCuSO₄-inducible vector for expression into the Drosophila S2 expressionsystem (DES; Invitrogen, San Diego, Calif., USA). Drosophila S2 cellswere transfected with 20 μg of the recombinant vector using the CalciumPhosphate Transfection kit according to the manufacturer's protocol(Invitrogen). Stable transfectants were established by cotransfectionwith 1 μg of pCoHygro vector and maintenance in the presence of 300μg/ml hygromycin. The expression of recombinant proteins was achievedusing copper sulfate at a final concentration of 500 μM. Culturesupernatants were collected 3 days after the induction, precipitatedwith 40% ammonium persulfate, and dialyzed against PBS.

Recombinant proteins were purified using a modified metal-ion affinitychromatography method as previously described. See, e.g., Lehr et al.,2000, Protein Expression Purif., 19: 362-68. Briefly, culturesupernatants or precipitated proteins were passed through a Pharmacia XK16 column packed with chelating sepharose fast flow (Pharmacia Biotech,Upsala, Sweden) and the recombinant proteins were eluted with 50 mMimidazole. Protein concentration was determined using Bradforddye-binding method or ELISA (R&D Systems, Minneapolis, Minn., USA).

(b) Characterization of CSA-CD40L Moieties by Western blot and ELISA

The expression of CSA-hCD40L was detected and quantified using theQuantikine CD40L immunoassay, which uses polyclonal Abs specific forCD40L pre-coated onto a microplate as described by manufacturer'sinstructions (R&D Systems). For Western blot analysis, supernatants ofCSA-hCD40L and CSA-mCD40L were first fractionated by sodium dodecylsulfate-polyacrylamide gel electrophoresis under native and denaturingconditions and then transferred onto polyvinylidene difluoride membranesusing a semidry-blot apparatus (BioRad, Hercules, Calif., USA).Membranes were first incubated in blocking buffer and then in goatanti-SA Ab (Pierce, Rockford, Ill., USA) at 1:1000 dilution in theblocking buffer for 1 hour at room temperature. Membranes were thenwashed extensively and incubated with horseradish peroxidase-conjugatedantigoat antibody at 1:4000 dilutions for 1 hour. Finally, the proteinswere detected using a chemiluminescent substrate according to themanufacturer's instructions (ECL, Amersham Biosciences, UK).

Transfectants expressed high levels of CSA-CD40L moieties that formedstable tetramers and higher order structures under nondenaturing PAGEconditions. Dissociation into monomers occurred only under denaturingconditions following heating at 100° C., but not 60° C. These datademonstrate that CD40L polypeptides of the immune co-stimulatorymoieties do not interfere with the expression, proper folding, andexistence of CSA as oligomers.

(c) Receptor Binding and Activation Assays

One million CD40 positive mouse A20 B cell lymphoma or human macrophageTHP-1 cells were incubated with 200 ng/ml of CSA-CD40L (human or mouse)moieties or control CSA protein at 4° C. for 30 min. After severalwashes with PBS, the bound proteins were detected using FITC conjugatedanti-streptavidin antibody (Vector Laboratories, Burlingame, Calif.,USA) in flow cytometry. CSA was used as a negative control to detectnonspecific binding. The effect of stimulation with CSA-CD40L on theexpression of CD80 and MHC class II molecules was determined byculturing 0.5×10⁶ THP-1 cells with 100 ng/ml of CSA-hCD40L or CSA orcoculturing with 0.5×10⁶ CHO cells transfected with the membrane form ofCD40L for 48 hours. Cells were then washed and stained with saturatingconcentrations of FITC conjugated anti-CD80 (L307.4) and HLA class II(TU36) antibodies (BD-PharMingen, San Diego, Calif., USA) and analyzedby flow cytometry.

CSA-mCD40L bound to both human and murine CD40 receptors (FIGS. 17A & B,dark line), as determined by flow cytometry and shown in FIG. 17. Incontrast, CSA-hCD40L interacted only with its receptor on human cells,with minimal to undetectable binding to murine cells (FIGS. 17C & D,dark line), demonstrating its species specificity. These interactionswere CD40-specific since there was no detectable binding when CSA usedas a control protein (FIG. 17, grey filled areas).

The upregulated expression of both MHC class II and CD80 molecules wasdetected on the surface of THP-1 cells using antibodies to HLA class II(FIG. 18A) and CD80 (FIG. 18B) molecules in flow cytometry at allCSA-hCD40L protein concentrations tested, with maximum upregulationachieved at 100 ng protein/5×10⁵ cells after 48 hours of stimulation.CSA-hCD40L (thin solid lines) was more effective than the membrane boundform of CD40L expressed on CHO cells (thick solid line) in upregulatingHLA class II molecules (MFI of 55.8 versus 35.5). In contrast, theupregulation of CD80 by both forms of CD40L was almost comparable (MFIof 33.6 versus 36.2). The upregulated expression was specific toCSA-hCD40L since incubation with CSA protein (solid histograms) did notsignificantly affect the expression of CD80 and HLA class II moleculesover background levels.

(d) Preparation of Bone Marrow-Derived DCs

Bone marrow was flushed from the femurs of 6- to 8-week old mice,dispersed into single cells by pipetting, and red blood cells were lysedwith ammonium chloride potassium (ACK) solution. The single cellsuspensions were then depleted for T and B cells using a cocktail of TIB105, TIB 146 and clone RL-172 hybridoma cell culture saturatedsupernatants for 30 minutes on ice. (Culture supernatants were a gift ofDr. Tatiana Zorina, University of Pittsburgh, Pa.). Cells were incubatedwith rabbit complement for 30 minutes at 37° C. and cultured overnight(37° C., 5% CO₂) in complete medium (RPMI 1640, 2 mM L-glutamine, 100μg/ml penicillin and streptomycin, 10% FBS, 0.1 mM nonessential aminoacids, 1 mM sodium pyruvate, 1 μg/ml indomethacine and 50 μMN-methyl-L-arginine) (Sigma) in six-well plates at a concentration of10⁶ cells/ml. Non-adherent cells were collected by gentle pipetting,counted, and resuspended at a concentration of 10⁵ cells/ml in completemedium supplemented with recombinant murine granulocyte-macrophagecolony-stimulating factor (5 ng/ml) and rmIL-4 (5 ng/ml) (All from USBiological, Swampscott, Mass., USA). Cells were cultured in six-wellplates (4 ml/well) for 5 days.

On the fifth day, DCs present in the culture were typed for theexpression of cell surface MHC and costimulatory molecules and incubatedwith varying concentrations (01-0.5 μg/10⁶ cells) of CA-mCD40L, mediumalone, or CSA. Cells were harvested on various days and analyzed for theexpression of maturation markers using PE-labeled monoclonal antibody(HL3) against CD11c and FITC labeled mAbs to CD80 (16-10A1) and CD86(GL1) (all from PharMingen).

The immature dendritic cells from day 5 murine bone marrow cultures thatwere incubated with various concentrations of mouse CSA-CD40L (01-0.5μg/10⁶ cells) for 48 hours showed increased expression of both CD80 andCD86 costimulatory molecules (FIGS. 19A & B), with the effect on theupregulation of CD80 expression greater than that for CD86 (3 versus 2fold) at 0.2 μg protein concentration per 10⁶ cells. Higherconcentrations of CSA-CD40L fusion proteins or longer incubation periodsdid not result in further upregulation (data not shown). This effect wasspecific to the immune co-stimulatory moiety because cells incubatedwith CSA protein had minimal to undetectable changes in the expressionof costimulatory molecules over background levels.

(e) Analysis of Pro-Inflammatory Cytokine Production

Human monocytes were plated in 96-well microtiter plates and stimulatedusing 1 μg/ml of a commercially available trimeric recombinant humanCD40L (rhsCD40L)+1 μg/ml enhancer (Alexis Biochemicals, San Diego,Calif., USA) and 100 ng/ml of CSA-hCD40L, CSA-mCD40L, or CSA.Supernatants were harvested after 18 hours of incubation and assayed byELISA using the OptEIA™ sets for all cytokines (PharMingen). Analysiswas performed using E-max Precision microplate reader (MolecularDevices, Sunnyvale, Calif., USA).

Ligation of human monocytes with CSA-hCD40L resulted in a 5-foldstimulation of human IL-1β production above CSA alone, which isequivalent to the levels induced by rhsCD40L (FIGS. 20A & B). Similarly,stimulation of human monocytes with CSA-mCD40L resulted in a robuststimulation of human IL-6 (FIGS. 20C & D). Thus, human and murineCSA-CD40L fusion proteins are both capable of stimulating CD40 on humanmonocytes to produce IL-β and IL-6.

(f) RNAse Protection Assay

Analysis of cytokine mRNA synthesis was performed by RNAse protectionassay. Cells were plated in a 6-well plate and stimulated via CD40 usingCSA-CD40L moieties for 3 to 4 hours. CHO transfectants expressing CD40Land rhsCD40L with enhancer were used as controls. RNA was extractedusing Trizol as described by manufacturer's instructions (Invitrogen).RNA (5 μg) was hybridized with a radiolabeled probe generated from thehuman cytokine/RNA template set, mCK-3b (RiboQuant, BD-PharMingen, SanDiego, Calif., USA), at 55° C. overnight. RNAse treatment was carriedout at 37° C. for 45 minutes, following which the protected probe waspurified and resolved by electrophoresis using a 5% polyacrylamide gel(BioRad) in TBE buffer. The gel was dried and exposed to Kodak Biomax XLX-ray film (Eastman Kodak, Rochester, N.Y., USA). With the undigestedprobe as markers, a standard curve was plotted as migration distancesversus nucleotide length on semi log paper. The identity of theRNAse-protected bands in the samples was then extrapolated from thegraph.

A shown in FIG. 20D, stimulation with CSA-hCD40L resulted in a 2.8-foldincrease in IL-6 mRNA over CSA alone.

Taken together, these data indicate that both human and murineCSA-CD40Ls are capable of inducing CD40 signaling in monocytes andmacrophages.

(g) CSA-hCD40L Stimulates iNOS Production in IFN-γ Primed Macrophages

CD40 ligation of IFN-γ primed macrophages results in the stimulation ofnitric oxide production, which plays a critical role in the microbicidaland cytotoxic activities of macrophages. Inducible nitric oxide synthase(iNOS) belongs to a family of nitric oxide synthases that catalyze thesynthesis of nitric oxide from L-arginine. Th1 and Th2 T helper cellscan differentially regulate arginine metabolism in macrophages. Th1cells induce iNOS production by macrophages while Th2 cells inducemacrophages to produce arginase which is associated withanti-inflammatory function. Thus, macrophage iNOS production is ahallmark of a Th1 type of immune response.

The ability of CSA-CD40L moieties to stimulate iNOS production in murinemacrophages was demonstrated as follows. CD40KO-human CD40 cells wereprimed for 24 hours with IFN-γ and subsequently stimulated withCSA-hCD40L, rhsCD40L, or CSA alone for 24 h. Cell lysates werenormalized and analyzed by Western blot using anti-iNOS Ab. Asdemonstrated in FIG. 21, stimulation of macrophages with CSA-hCD40L orrhsCD40L, but not CSA, resulted in the stimulation of iNOS production.Stimulation with 1 μg/ml of commercial rhsCD40L resulted in a 6-foldstimulation of iNOS above background, while stimulation with 300 ng/mlof CSA-hCD40L resulted in a 9-fold stimulation of iNOS above CSA alone.These data indicate that CSA-hCD40L is a potent stimulator of macrophageiNOS production.

Example 20 In Vivo Killing Response Induced by CSA-4-1BBL

Naïve C57BL/6 mice were immunized intravenously with 50 μg ovalbumin(OVA) as the antigen, and two doses (12.5 μg and 25 μg, respectively) ofCSA-4-1BBL or LPS as an adjuvant. Naïve animals were used as a control.

Seven days later, all mice received CFSE labeled target cells., whichwere prepared as follows. Splenocytes from naïve C57BL/6 mice weredivided into two populations. The first population was labeled with 0.25μM CFSE (CFSE^(low)) and the second population was labeled with 2.5 μMCFSE and then pulsed with 2 μg/ml OVA₂₅₇₋₂₆₄ peptide (SIINFEKL) for 1hour (CFSE^(hi)). Cells were mixed at a ratio of 1:1 ad a total of 1×10⁷cells were injected intravenously into recipient animals. Spleens wereharvested 48 hours later, and CSFE fluorescence intensity was analyzedby flow cytometry. Results are shown in FIG. 22, expressed as thepercentage lysis of the peptide-pulsed CFSE^(hi) peak as compared to thereference CFSE^(low) peak, normalized to naïve animals. As shown in FIG.22, immunization with OVA and CSA-4-1BBL generated a potent in vivokilling response in target cells, and CSA-4-1BBL demonstrated a strongeradjuvant effect than LPS at both concentrations tested.

Example 21 Costimulation with 4-1BBL Greatly Enhanced the ImmuneResponse to HPV16 E7 Protein in Mice, Controlled TC-1 Tumors, andInduced Anti-Tumor Memory

Naïve B6 mice were challenged subcutaneously in the right flank with1×10⁵ live TC-1 cells which stably express the human papillomavirus-16E7 protein, in a vaccination protocol based on the administration of theCD8+ T cell epitope of the HPV16 E7 epitope P1 (having the amino acidsequence RAHYNIVTF). FIG. 23 shows survival of mice in this TC-1 tumormodel. After 10 days, mice received one subcutaneous injection of either(i) PBS (♦, n=20); (ii) 50 μg P1+12.5 μg CSA (, n=6) (iii) 25 μgCSA-4-1BBL (▴, n=10); (iv) 50 μg P1+25 μg CSA-4-1BBL (Δ, n=13), or (v)50 μg P1+10 μg CpG (□, n=7).

As shown in FIG. 23, immunization with P1 or CSA-4-1BBL achieved somesuccessful immunotherapy, but better results (including enhancedsurvival) were achieved by immunization with both P1 and CSA-4-1BBL. Allanimals receiving only PBS developed tumors.

The surviving animals were re-challenged at day 60 (black arrow). Tumorgrowth was monitored 3 times a week. Administration of CSA-4-1BBL and P1together after the tumor challenge significantly increased the survivalof animals compared to P1 or CSA-4-1BBL alone, or P1 and CpG.Importantly, none of the surviving animals in the P1+CSA-4-1BBL groupdeveloped tumor upon secondary challenge, demonstrating immunologicalmemory.

Example 22 Vaccination with OVA/CSA-4-1BBL Prevents Tumor Growth

Naïve C57BL/6 mice were immunized with 50 μg OVA or 50 μg biotinylatedOVA conjugated to 25 μg CSA-4-1BBL. Some animals were left untreated ascontrols. After 7 days, mice were challenged subcutaneously in the rightflank with 1×10⁵ OVA-expressing EG.7 tumor cells. Tumor growth wasmonitored three times a week using calipers. The results (tumor-freesurvival) are shown in FIG. 24. As shown, all control animals andanimals vaccinated with OVA developed tumors, while all animalsvaccinated with biotinylated OVA/CSA-4-1BBL did not develop tumors,demonstrating that vaccination with biotinylated OVA/CSA-4-1BBL resultedin 100% prevention of the growth of thyoma tumors.

Example 23 4-1BBL Strongly Enhances the Antigen-Specific CTL Response InVivo

Naïve C57BL/6 mice were immunized intravenously with (i) 50 μg OVA, (ii)50 μg OVA and 25 μg CSA-4-1BBL, (iii) 50 μg OVA and 25 μg anti-CD137antibody or (iv) 50 μg OVA and 25 μg LPS. Naïve animals were used ascontrol. Seven days later, all mice received CFSE labeled target cells.Briefly, splenocytes from naïve C57BL/6 were divided into twopopulation. The first population was labeled with 0.25 μM CFSE(CFSElow). The second population was labeled with 2.5 μM CFSE and thenpulsed with 2 μg/ml OVA₂₅₇₋₂₆₄ SIINFEKL peptide for 1 hour (CFSEhi).Cells were mixed at a ratio of 1:1 and a total of 1×10⁷ cells wereinjected intravenously into recipient animals. Spleens were harvested 48hours later and CFSE fluorescence intensity was analyzed by flowcytometry, with the results shown in FIG. 25. The results are expressedon the corner of each panel as percentage lysis of the peptide pulsedCFSEhi peak as compared with the reference CFSElow peak normalized tonaïve animal. This assay revealed that 4-1BBL could enhance the antigenspecific CTL response to higher levels (95%) compared to antigen (OVA)alone (24.2%), or antigen and LPS (35%), resulting in killing ofmajority of target cells.

Example 24 4-1BBL Costimulation Increases Antigen Presentation to CD8+T-Cells In Vivo

Naïve B6-SJL (CD45.1+) animals were immunized intravenously with (i) 10μg OVA, (ii) 10 μg OVA and 5 μg 4-1BBL, or (iii) left untreated. After 2days, animals received 1×10⁶ CFSE labeled OT-1 cells (CD45.2+) byintravenous injection. Spleen were harvested 3 days later andproliferation of OT-1 cells were analyzed using flow cytometry, as showsin FIG. 26. Administration of 4-1 BBL together with the antigenincreased the antigen presentation to CD8+ T cells as demonstrated byproliferation of majority of OT-1 cells. (83.2% for OVA+4-1BBL; 13.7%for OVA; 8.8% for no treatment).

Example 25 4-1BBL Costimulation Increases Antigen Uptake by DendriticCells

Naïve BALB/c mice were injected subcutaneously with 25 μg OVA-FITC, 25μg OVA-FITC and 10 μg CSA, or 25 μg OVA-FITC and 25 μg CSA-4-1BBL. After3 hours, ingunial lymph nodes at the site of injection were harvested.FITC+ cells in CD11c+population was analyzed using flow cytometry todetermine in vivo fluorescently-labelled antigen update, as seen in FIG.27. As shown, 4-1BBL signaling increased the antigen uptake by CD11c+DCs whereas the control CSA protein had no effect.

While the invention has been described and exemplified in sufficientdetail for those skilled in this art to make and use it, variousalternatives, modifications, and improvements should be apparent withoutdeparting from the spirit and scope of the invention. The examplesprovided herein are representative of preferred embodiments, areexemplary, and are not intended as limitations on the scope of theinvention. Modifications therein and other uses will occur to thoseskilled in the art. These modifications are encompassed within thespirit of the invention and are defined by the scope of the claims.

It will be readily apparent to a person skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.

All patents and publications mentioned in the specification areindicative of the levels of those of ordinary skill in the art to whichthe invention pertains. All patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments and optional features, modification and variationof the concepts herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

Other exemplary embodiments are set forth below and in the claims thatfollow:

1. A combination comprising: (a) a first conjugate comprising (i) aconjugate member comprising a first immune co-stimulatory polypeptideand (ii) a conjugate member comprising a first member of a binding pair;and (b) a second conjugate comprising (i) a conjugate member comprisinga first antigen and (ii) a conjugate member comprising a second memberof said binding pair.
 2. The combination of embodiment 1, wherein saidfirst member of said binding pair comprises avidin or streptavidin andsaid second member of said binding pair comprises biotin.
 3. Thecombination of embodiment 2, wherein said first member of said bindingpair comprises core streptavidin.
 4. The combination of embodiment 1,wherein said first conjugate comprises a fusion polypeptide comprisingsaid first immune co-stimulatory polypeptide and said first member ofsaid binding pair.
 5. The combination of embodiment 1, wherein saidfirst immune co-stimulatory polypeptide is selected from the groupconsisting of 4-1BBL, CD86, ICOSL, PD-L1, PD-L2, B7-H3, B7-H4, OX40L,CD27L, CD30L, LIGHT, BAFF, APRIL, CD80 and CD40L.
 6. The combination ofembodiment 5, wherein said first immune co-stimulatory polypeptide is4-1BBL.
 7. The combination of embodiment 6, wherein said first conjugatecomprises a fusion polypeptide comprising the amino acid sequence of SEQID NO:8.
 8. The combination of embodiment 1, wherein said first antigenis associated with an infectious agent.
 9. The combination of embodiment8, wherein said infectious agent is selected from the group consistingof human or avian influenza and human immunodeficiency virus.
 10. Thecombination of embodiment 1, wherein said first antigen is a tumorassociated antigen.
 11. The combination of embodiment 10, wherein saidtumor associated antigen is selected from the group consisting of humantelomerase reverse transcriptase, survivin, MAGE-1, MAGE-3, humanchorionic gonadotropin, carcinoembryonic antigen, alpha fetoprotein,pancreatic oncofetal antigen, MUC-1, CA 125, CA 15-3, CA 19-9, CA 549,CA 195, prostate-specific antigens; prostate-specific membrane antigen,Her2/neu, gp-100, mutant K-ras proteins, mutant p53, truncated epidermalgrowth factor receptor, chimeric protein ^(p210)BCR-ABL; HPV E6, HPV E7;Epstein-Barr virus EBNA3 protein, and mixtures or fragments thereof. 12.The combination of embodiment 1, wherein said first and secondconjugates are provided as separate compositions.
 13. The combination ofembodiment 1, wherein said first and second conjugates are provided as asingle composition.
 14. The combination of embodiment 13, wherein saidcomposition comprises a pharmaceutically acceptable carrier, excipientor diluent.
 15. The combination of embodiment 13, wherein, as providedin said composition, said first conjugate is bound to said secondconjugate via binding between said first and second binding pairmembers.
 16. The combination of embodiment 1, wherein said first immuneco-stimulatory polypeptide does not comprise the transmembrane domain ofan immune co-stimulatory molecule.
 17. The combination of embodiment 1,wherein said first immune co-stimulatory polypeptide comprises theextracellular domain of an immune co-stimulatory molecule, or a receptorbinding portion thereof.
 18. The combination of embodiment 1, furthercomprising a third conjugate comprising (i) a conjugate membercomprising a second immune co-stimulatory polypeptide and a first memberof a binding pair and (ii) a conjugate member comprising a secondantigen and a second member of a binding pair, wherein: said secondimmune co-stimulatory polypeptide is the same as or different from saidfirst immune co-stimulatory polypeptide; said second antigen is the sameas or different from said first antigen; said first and second bindingpair members of said third conjugate are the same as or different fromsaid first and second binding pair members of said first and secondconjugates, and said first conjugate member is bound to said secondconjugate member via binding between said first and second binding pairmembers.
 19. A combination comprising: (a) a first conjugate comprising(i) a conjugate member comprising a first immune co-stimulatorypolypeptide and (ii) a conjugate member comprising a first member of abinding pair; and (b) a second conjugate comprising (i) a conjugatemember comprising an infectious agent and (ii) a conjugate membercomprising a second member of said binding pair.
 20. A method ofgenerating or enhancing an immune response against a tumor whichexpresses a first tumor-associated antigen, comprising administering toa patient with said tumor: (a) a first conjugate comprising (i) aconjugate member comprising a first immune co-stimulatory polypeptideand (ii) a conjugate member comprising a first member of a binding pair,and a second conjugate comprising (i) a conjugate member comprising saidfirst tumor-associated antigen and (ii) a conjugate member comprising asecond member of said binding pair; or (b) immune cells which have beentreated in vitro with said first and second conjugates.
 21. The methodof embodiment 20, wherein said first and second conjugates areadministered to said patient.
 22. The method of embodiment 21, whereinsaid first and second conjugates are administered separately.
 23. Themethod of embodiment 21, wherein said first and second conjugates areadministered simultaneously.
 24. The method of embodiment 20, whereinsaid first and second conjugates are provided in a single composition.25. The method of embodiment 24, wherein, as provided in saidcomposition, said first conjugate is bound to said second conjugate viabinding between said first and second binding pair members.
 26. Themethod of embodiment 21, wherein at least one of said first and secondconjugates is administered by intratumoral injection.
 27. The method ofembodiment 20, wherein said first tumor-associated antigen is selectedfrom the group consisting of human telomerase reverse transcriptase,survivin, MAGE-1, MAGE-3, human chorionic gonadotropin, carcinoembryonicantigen, alpha fetoprotein, pancreatic oncofetal antigen, MUC-1, CA 125,CA 15-3, CA 19-9, CA 549, CA 195, prostate-specific antigens;prostate-specific membrane antigen, Her2/neu, gp-100, mutant K-rasproteins, mutant p53, truncated epidermal growth factor receptor,chimeric protein ^(p210)BCR-ABL; HPV E6, HPV E7; Epstein-Barr virusEBNA3 protein, and mixtures or fragments thereof.
 28. The method ofembodiment 20, further comprising administering a third conjugatecomprising (i) a conjugate member comprising a second immuneco-stimulatory polypeptide and a first member of a binding pair and (ii)a conjugate member comprising a second tumor-associated antigen and asecond member of a binding pair, wherein: said second immuneco-stimulatory polypeptide is the same as or different from said firstimmune co-stimulatory polypeptide; said second antigen is the same as ordifferent from said first antigen; said first and second binding pairmembers of said third conjugate are the same as or different from saidfirst and second binding pair members of said first and secondconjugates, and said first conjugate member is bound to said secondconjugate member via binding between said first and second binding pairmembers.
 29. The method of embodiment 28, wherein said secondtumor-associated antigen is selected from the group consisting of humantelomerase reverse transcriptase, survivin, MAGE-1, MAGE-3, humanchorionic gonadotropin, carcinoembryonic antigen, alpha fetoprotein,pancreatic oncofetal antigen, MUC-1, CA 125, CA 15-3, CA 19-9, CA 549,CA 195, prostate-specific antigens; prostate-specific membrane antigen,Her2/neu, gp-100, mutant K-ras proteins, mutant p53, truncated epidermalgrowth factor receptor, chimeric protein ^(p210)BCR-ABL; HPV E6, HPV E7;Epstein-Barr virus EBNA3 protein, and mixtures or fragments thereof. 30.The method of embodiment 20, wherein said first member of said bindingpair comprises avidin or streptavidin and said second member of saidbinding pair comprises biotin.
 31. The method of embodiment 30, whereinsaid first member of said binding pair comprises core streptavidin. 32.The method of embodiment 20, wherein said first conjugate comprises afusion polypeptide comprising said first immune co-stimulatorypolypeptide and said first member of said binding pair.
 33. The methodof embodiment 20, wherein said first immune co-stimulatory polypeptideis selected from the group consisting of 4-1BBL, CD86, ICOSL, PD-L1,PD-L2, B7-H3, B7-H4, OX40L, CD27L, CD30L, LIGHT, BAFF, APRIL, CD80 andCD40L.
 34. The method of embodiment 33, wherein said first immuneco-stimulatory polypeptide is 4-1BBL.
 35. The method of embodiment 34,wherein said first conjugate comprises a fusion polypeptide comprisingthe amino acid sequence of SEQ ID NO:8.
 36. The method of embodiment 20,wherein said immune co-stimulatory polypeptide does not comprise atransmembrane domain of an immune co-stimulatory molecule.
 37. Themethod of embodiment 20, wherein said immune co-stimulatory polypeptidecomprises the extracellular domain of an immune co-stimulatory molecule,or a receptor binding portion thereof.
 38. The method of embodiment 20,wherein said patient is administered immune cells which have beentreated in vitro with said first and second conjugates.
 39. The methodof embodiment 38, wherein said immune cells comprise a receptor for saidimmune co-stimulatory polypeptide, and wherein said first conjugate isconjugated to said immune cells via binding between said immuneco-stimulatory polypeptide and said receptors, and said second conjugateis conjugated to said immune cells via binding between said first andsecond binding pair members.
 40. The method of embodiment 38, whereinsaid immune cells are treated with said first and second conjugatessimultaneously.
 41. The method of embodiment 38, wherein said immunecells are treated with said first and second conjugates separately. 42.A method of modifying immune cells to generate or enhance an immuneresponse to a tumor expressing a tumor-associated antigen or to aninfectious agent, comprising contacting immune cells expressing areceptor for a first immune co-stimulatory polypeptide with: (a) a firstconjugate comprising (i) a conjugate member comprising said first immuneco-stimulatory polypeptide and (ii) a conjugate member comprising afirst member of a binding pair; and (b) a second conjugate comprising(i) a conjugate member comprising an antigen associated with said tumoror infectious agent or said infectious agent and (ii) a conjugate membercomprising a second member of said binding pair, wherein said firstconjugate is conjugated to said immune cells via binding between saidimmune co-stimulatory polypeptide and said receptor, and said secondconjugate is conjugated to said immune cell via binding between saidfirst and second binding pair members.
 43. The method of embodiment 42,wherein said first conjugate and second conjugates are contactedseparately.
 44. The method of embodiment 42, wherein said first andsecond conjugates are contacted simultaneously.
 45. The method ofembodiment 44, wherein said first and second conjugates are provided ina single composition.
 46. The method of embodiment 45, wherein, asprovided in said composition, said first conjugate is bound to saidsecond conjugate via binding between said first and second binding pairmembers.
 47. The method of embodiment 42, wherein said contacting iseffected by administering said first and second conjugates to a patientcontaining said immune cells.
 48. The method of embodiment 47, whereinsaid second conjugate comprises a tumor associated antigen, said patientfurther comprises said tumor, and at least one of said first and secondconjugates is administered by intratumoral injection.
 49. The method ofembodiment 42, wherein said immune cell is a T cell or neutrophil. 50.The method of embodiment 49, wherein said T cell is selected from thegroup consisting of CD4+ cells, CD8+ cells, natural killer cells,monocytes and dendritic cells.
 51. The method of embodiment 42, whereinsaid second conjugate comprises a tumor-associated antigen.
 52. Themethod of embodiment 51, wherein said tumor-associated antigen isselected from the group consisting of human telomerase reversetranscriptase, survivin, MAGE-1, MAGE-3, human chorionic gonadotropin,carcinoembryonic antigen, alpha fetoprotein, pancreatic oncofetalantigen, MUC-1, CA 125, CA 15-3, CA 19-9, CA 549, CA 195,prostate-specific antigens; prostate-specific membrane antigen,Her2/neu, gp-100, mutant K-ras proteins, mutant p53, truncated epidermalgrowth factor receptor, chimeric protein ^(p210)BCR-ABL; HPV E6, HPV E7;Epstein-Barr virus EBNA3 protein, and mixtures or fragments thereof. 53.The method of embodiment 42, wherein said second conjugate comprises anantigen associated with an infectious agent or the infectious agent. 54.The method of embodiment 42, wherein said infectious agent is abacteria.
 55. The method of embodiment 54, wherein said bacteria isselected from the group consisting of Mycobacterium tuberculosis;Bacillus anthracis; Staphylococcus aureus.
 56. The method of embodiment42, wherein said infectious agent is a virus.
 57. The method ofembodiment 56, wherein said virus is selected from the group consistingof Adenoviridae; Arenaviridae Caliciviridae; Coronaviridae; Filoviridae;Flaviviridae; Hepadnaviridae; Herpesviridae; Orthomyxoviridae;Papillomaviridae; Picornaviridae; Poxyiridae; Reoviridae; Retroviridae;Rhabdoviridae; and Togaviridae;
 58. The method of embodiment 42, whereinsaid infectious agent is a parasite
 59. The method of embodiment 58,wherein said parasite is selected from the group consisting ofPlasmodium and Leishmania.
 60. The method of embodiment 42, wherein saidinfectious agent is a fungus
 61. The method of embodiment 60, whereinsaid fungus is selected from the group consisting of Aspergillis;Candida; Coccidia; Cryptococci; Geotricha; Histoplasma; Microsporidia;and Pneumocystis
 62. The method of embodiment 47, wherein said patientis selected from the group consisting of equine, ovine, caprine, bovine,porcine, avian, canine, feline and primate species.
 63. The method ofembodiment 47, wherein said patient is human.
 64. The method ofembodiment 42, wherein said immune cells comprise a receptor for asecond immune co-stimulatory polypeptide, the method further comprisingcontacting said immune cells with a third conjugate comprising (i) aconjugate member comprising said second immune co-stimulatorypolypeptide and a first member of a binding pair and (ii) a conjugatemember comprising a second antigen associated with said tumor orinfectious agent or said infectious agent and a second member of saidbinding pair, wherein: said second immune co-stimulatory polypeptide isthe same as or different from said first immune co-stimulatorypolypeptide; said second antigen, if present, is the same as ordifferent from said first antigen, if present; said first and secondbinding pair members of said third conjugate are the same as ordifferent from said first and second binding pair members of said firstand second conjugates, and said first conjugate member is bound to saidsecond conjugate member via binding between said first and secondbinding pair members.
 65. The method of embodiment 42, wherein saidfirst member of said binding pair comprises avidin or streptavidin andsaid second member of said binding pair comprises biotin.
 66. The methodof embodiment 65, wherein said first member of said binding paircomprises core streptavidin.
 67. The method of embodiment 42, whereinsaid first conjugate comprises a fusion polypeptide comprising saidfirst immune co-stimulatory polypeptide and said first member of saidbinding pair.
 68. The method of embodiment 42, wherein said first immuneco-stimulatory polypeptide is selected from the group consisting of4-1BBL, CD86, ICOSL, PD-L1, PD-L2, B7-H3, B7-H4, OX40L, CD27L, CD30L,LIGHT, BAFF, APRIL, CD80 and CD40L.
 69. The method of embodiment 68,wherein said first immune co-stimulatory polypeptide is 4-1BBL.
 70. Themethod of embodiment 69, wherein said first conjugate comprises a fusionpolypeptide comprising the amino acid sequence of SEQ ID NO:8.
 71. Themethod of embodiment 42, wherein said immune co-stimulatory polypeptidedoes not comprise a transmembrane domain of an immune co-stimulatorymolecule.
 72. The method of embodiment 42, wherein said immuneco-stimulatory polypeptide comprises the extracellular domain of animmune co-stimulatory molecule, or a receptor binding portion thereof.73. A population of immune cells made by the method of embodiment 42,wherein said immune cells generate or enhance an immune response to saidtumor when contacted with other immune cells.
 74. A modified immune cellexpressing a receptor for a first immune co-stimulatory polypeptide,wherein said modified immune cell comprises: a) a first conjugatecomprising (i) a conjugate member comprising said first immuneco-stimulatory polypeptide and (ii) a conjugate member comprising afirst member of a binding pair; and (b) a second conjugate comprising(i) a conjugate member comprising a first antigen or infectious agentand (ii) a conjugate member comprising a second member of said bindingpair, wherein said first conjugate is conjugated to said immune cell viabinding between said immune co-stimulatory polypeptide and saidreceptor, and said second conjugate is conjugated to said immune cellvia binding between said first and second binding pair members.
 75. Theimmune cell of embodiment 74, wherein said immune cell is selected fromthe group consisting of T cells, neutrophils, natural killer cells,monocytes and dendritic cells.
 76. The immune cell of embodiment 75,wherein said T cell is selected from the group consisting of CD4+ cellsand CD+ cells.
 77. The immune cell of embodiment 76, wherein said firstmember of said binding pair comprises avidin or streptavidin and saidsecond member of said binding pair comprises biotin.
 78. The immune cellof embodiment 76, wherein said first member of said binding paircomprises core streptavidin.
 79. The immune cell of embodiment 74,wherein said first conjugate comprises a fusion polypeptide comprisingsaid first immune co-stimulatory polypeptide and said first member ofsaid binding pair.
 80. The immune cell of embodiment 74, wherein saidfirst immune co-stimulatory polypeptide is selected from the groupconsisting of 4-1BBL, CD86, ICOSL, PD-L1, PD-L2, B7-H3, B7-H4, OX40L,CD27L, CD30L, LIGHT, BAFF, APRIL, CD80 and CD40L.
 81. The immune cell ofembodiment 80, wherein said first immune co-stimulatory polypeptide is4-1BBL.
 82. A method of inducing or enhancing an immune response againstan infectious agent, comprising administering to a patient sufferingfrom or at risk of infection with said infectious agent: a) a firstconjugate comprising (i) a conjugate member comprising a first immuneco-stimulatory polypeptide and (ii) a conjugate member comprising afirst member of a binding pair; and (b) a second conjugate comprising(i) a conjugate member comprising a first antigen associated with saidinfectious agent or comprising said infectious agent and (ii) aconjugate member comprising a second member of said binding pair. 83.The method of embodiment 82, wherein said first and second conjugatesare administered separately.
 84. The method of embodiment 82, whereinsaid first and second conjugates are administered simultaneously. 85.The method of embodiment 84, wherein said first and second conjugatesare provided in a single composition.
 86. The method of embodiment 85,wherein, as provided in said composition, said first conjugate is boundto said second conjugate via binding between said first and secondbinding pair members.
 87. The method of embodiment 82, wherein at leastone of said first and second conjugates is administered by a routeselected from the group consisting of: oral; sublingual; transmucosal;transdermal; rectal; vaginal; subcutaneous; intramuscular; intravenous;intra-arterial; intrathecal; via catheter; via implant; and directlyinto a tumor.
 88. The method of embodiment 82, wherein said infectiousagent is a bacteria.
 89. The method of embodiment 88, wherein saidbacteria is selected from the group consisting of Mycobacteriumtuberculosis; Bacillus anthracis; Staphylococcus aureus.
 90. The methodof embodiment 82, wherein said infectious agent is a virus.
 91. Themethod of embodiment 90, wherein said virus is selected from the groupconsisting of Adenoviridae; Arenaviridae Caliciviridae; Coronaviridae;Filoviridae; Flaviviridae; Hepadnaviridae; Herpesviridae;Orthomyxoviridae; Papillomaviridae; Picornaviridae; Poxyiridae;Reoviridae; Retroviridae; Rhabdoviridae; and Togaviridae;
 92. The methodof embodiment 82, wherein said infectious agent is a parasite
 93. Themethod of embodiment 92, wherein said parasite is selected from thegroup consisting of Plasmodium and Leishmania.
 94. The method ofembodiment 82, wherein said infectious agent is a fungus
 95. The methodof embodiment 94, wherein said fungus is selected from the groupconsisting of Aspergillis; Candida; Coccidia; Cryptococci; Geotricha;Histoplasma; Microsporidia; and Pneumocystis
 96. The method ofembodiment 82, wherein said patient is selected from the groupconsisting of equine, ovine, caprine, bovine, porcine, avian, canine,feline and primate species.
 97. The method of embodiment 96, whereinsaid patient is human.
 98. The method of embodiment 82, wherein saidinfection is human or avian influenza and said first antigen is selectedfrom the group consisting of H, N, M1, M2e, NS1, NS2 (NEP), NP, PA, PB1,and PB2.
 99. The method of embodiment 82, wherein said infection is HIVand said first antigen is selected from the group of HIV antigensconsisting of Gag proteins, Pol, Vif, Vpr, Rev, Vpu, envelope eptiopes,Tat, and Nef.
 100. The method of embodiment 82, further comprisingadministering a third conjugate comprising (i) a conjugate membercomprising a second immune co-stimulatory polypeptide and a first memberof a binding pair and (ii) a conjugate member comprising a secondantigen associated with said infection or said infectious agent and asecond member of said binding pair, wherein: said second immuneco-stimulatory polypeptide is the same as or different from said firstimmune co-stimulatory polypeptide; said second antigen, if present, isthe same as or different from said first antigen, if present; said firstand second binding pair members of said third conjugate are the same asor different from said first and second binding pair members of saidfirst and second conjugates, and said first conjugate member is bound tosaid second conjugate member via binding between said first and secondbinding pair members.
 101. The method of embodiment 100, wherein saidinfection is human or avian influenza and said second antigen isselected from the group consisting of H, N, M1, M2e, NS1, NS2 (NEP), NP,PA, PB1, and PB2.
 102. The method of embodiment 101, wherein saidinfection is HIV and said second antigen is selected from the group ofHIV antigens consisting of Gag proteins, Pol, Vif, Vpr, Rev, Vpu,envelope eptiopes, Tat, and Nef.
 103. The method of embodiment 82,wherein said first member of said binding pair comprises avidin orstreptavidin and said second member of said binding pair comprisesbiotin.
 104. The method of embodiment 103, wherein said first member ofsaid binding pair comprises core streptavidin.
 105. The method ofembodiment 82, wherein said first conjugate comprises a fusionpolypeptide comprising said first immune co-stimulatory polypeptide andsaid first member of said binding pair.
 106. The method of embodiment82, wherein said first immune co-stimulatory polypeptide is selectedfrom the group consisting of 4-1BBL, CD86, ICOSL, PD-L1, PD-L2, B7-H3,B7-H4, OX40L, CD27L, CD30L, LIGHT, BAFF, APRIL, CD80 and CD40L.
 107. Themethod of embodiment 106, wherein said first immune co-stimulatorypolypeptide is 4-1BBL.
 108. The method of embodiment 107, wherein saidfirst conjugate comprises a fusion polypeptide comprising the amino acidsequence of SEQ ID NO:8.
 109. The method of embodiment 82, wherein saidimmune co-stimulatory polypeptide does not comprise a transmembranedomain of an immune co-stimulatory molecule.
 110. The method ofembodiment 82, wherein said immune co-stimulatory polypeptide comprisesthe extracellular domain of an immune co-stimulatory molecule, or areceptor binding portion thereof.
 111. A conjugate consistingessentially of an immune co-stimulatory polypeptide and avidin orstreptavidin, wherein said immune co-stimulatory polypeptide is selectedfrom the group consisting of 4-1BBL, CD86, ICOSL, PD-L1, PD-L2, B7-H3,B7-H4, OX40L, CD27L, CD30L, LIGHT, BAFF, APRIL, CD80 and CD40L.
 112. Theconjugate of embodiment 111, comprising core streptavidin.
 113. Theconjugate of embodiment 111, wherein said immune co-stimulatorypolypeptide is selected from the group consisting of 4-1 BBL, ICOSL,PD-L1, PD-L2, OX40L, CD27L, CD30L, LIGHT, BAFF, and APRIL.
 114. A methodof inducing an immunostimulatory response in an animal consistingessentially of administering to the animal a conjugate consistingessentially of an immune co-stimulatory polypeptide and avidin orstreptavidin.
 115. The method of embodiment 114, wherein said conjugatecomprises core streptavidin.
 116. The method of embodiment 114, whereinsaid immune co-stimulatory polypeptide is selected from the groupconsisting of 4-1BBL, CD86, ICOSL, PD-L1, PD-L2, B7-H3, B7-H4, OX40L,CD27L, CD30L, LIGHT, BAFF, APRIL, CD80 and CD40L.
 117. The method ofembodiment 116, wherein said immune co-stimulatory polypeptide isselected from the group consisting of 4-1 BBL, ICOSL, PD-L1, PD-L2,OX40L, CD27L, CD30L, LIGHT, BAFF, and APRIL.
 118. A conjugate comprisingan immune co-stimulatory polypeptide and avidin or streptavidin, whereinsaid immune co-stimulatory polypeptide is selected from the groupconsisting of 4-1BBL, CD86, ICOSL, PD-L1, PD-L2, B7-H3, B7-H4, OX40L,CD27L, CD30L, LIGHT, BAFF, APRIL, CD80 and CD40L.
 119. A method ofinducing an immunostimulatory response in an animal comprisingadministering to the animal a conjugate comprising an immuneco-stimulatory polypeptide and avidin or streptavidin, wherein saidimmune co-stimulatory polypeptide is selected from the group consistingof 4-1BBL, CD86, ICOSL, PD-L1, PD-L2, B7-H3, B7-H4, OX40L, CD27L, CD30L,LIGHT, BAFF, APRIL, CD80 and CD40L.
 120. The method of embodiment 119,further comprising administering an antigen to the animal.
 121. Themethod of embodiment 120, wherein said antigen is administered as aconjugate comprising said antigen and a member of a binding pair.